Treatment of existing left ventricular heart failure

ABSTRACT

The present disclosure provides methods of treating diuretic resistance by administering an IL-6 antagonist to patients who require diuresis. In typical embodiments, the patient has heart failure. Optionally, the patient has elevated urine levels of IL-6, plasma levels of IL-6, or both urine and plasma levels of IL-6.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national stage filing ofInternational Application No. PCT/US2018/016508, filed on Feb. 1, 2018,which claims priority to U.S. Application No. 62/453,257 filed on Feb.1, 2017. The contents of the aforementioned applications are herebyincorporated by reference in their entireties.

1. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grants5R01HL128973 and 4K23HL114868 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

3. STATEMENT REGARDING JOINT RESEARCH AGREEMENT

This invention was made under a Joint Research Agreement by and amongYale University, MedImmune Ltd., AstraZeneca Pharmaceuticals LP, andCorvidia Therapeutics, Inc.

4. SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated herein by reference inits entirety. Said ASCII copy, created on Feb. 9, 2018, is named35789WO_CRF_sequencelisting.txt, and is 46, 377 bytes in size.

5. BACKGROUND

Renal injury is often observed in heart failure, and heart failure isoften observed in renal disease. The term cardiorenal syndrome (“CRS”)encompasses a variety of clinical conditions in which dysfunction of theheart, kidney, or both, leads to accelerated failure of both organs.Evidence of this adverse organ crosstalk portends a high degree ofmorbidity and mortality. Despite its dire clinical implications, themechanistic underpinnings of CRS are only now being elucidated.

There is a need for new methods of treating renal injury and renalimpairment in heart failure patients. There is also a need for newmethods of detecting cardiorenal syndrome, for determining whichcardiorenal patients will be responsive to treatment, and for monitoringefficacy of therapy.

Diuretics are a mainstay in the treatment of heart failure. However,certain patients are, or become, resistant to diuretics. There is a needfor new methods of treating patients who require diuretics but areresistant to diuretics.

6. SUMMARY

As further described below in Example 1, consecutive heart failure(“HF”) patients receiving high dose diuretic therapy at an outpatienttreatment center were enrolled in a prospective observational study.Plasma levels of IL-6 were measured to query systemic associations ofthis pro-inflammatory cytokine with various disease parameters, andurine levels of IL-6 were measured to query IL-6 associations with localinflammation and neurohormonal activation at the level of renal tissues.

Urine IL-6 and plasma IL-6 levels were found to be only modestlycorrelated with one another.

Increases in urine levels of IL-6 were significantly correlated in theseheart failure patients with measures of renal impairment, such asdiuretic resistance, lower estimated glomerular filtration rate(“eGFR”), and increased tissue-level renin-angiotensin-aldosteronesystem (“RAAS”) activation.

Although an inverse association between diuretic efficiency and plasmaIL-6 was also observed, upon adjustment for eGFR, only urine IL-6remained significantly associated with risk of low diuretic efficiencyin these patients. Furthermore, when urine IL-6 and plasma IL-6 wereboth entered into a logistic regression model, only urine IL-6 remainedassociated with risk of low diuretic efficiency while plasma IL-6 showedno such association.

These data demonstrate that urine IL-6 level is a useful biomarker forrenal inflammation, and can be used to gauge renal dysfunction in thesetting of heart failure (cardiorenal syndrome). The data furthersuggest that serial measurements of urine IL-6 can be used to measurethe renal benefits of treatments administered to patients with heartfailure, notably heart failure patients with cardiorenal syndrome.

The urine IL-6 data, and to some extent the plasma IL-6 data, alsopredict that treatment with an IL 6 antagonist should be effective toreduce renal inflammation in heart failure patients, that is, to treatrenal symptoms of cardiorenal syndrome.

However, because infection is often a precipitating cause of acutedecompensation in heart failure patients, it is important to limitanti-cytokine and other immunosuppressive therapies to those heartfailure patients who are likely to respond with improved renal and/orcardiac function. The cost of chronic IL-6 antagonist therapy alsomilitates for limiting treatment to those heart failure patients who arelikely to respond with improved renal and/or cardiac function.

Analysis was expanded to 129 patients, and further assessed eachpatient's genotype at the rs855791 single nucleotide polymorphism(“SNP”) in transmembrane protease serine 6 (“TMPRSS6”).

Urine levels of IL-6 were inversely correlated with diuretic efficiencyonly in the patients having at least one copy of the major allele of theTMPRSS6 rs855791 SNP (AG and GG); urine levels of IL-6 were notsignificantly correlated with diuretic efficiency in patients homozygousfor the minor allele (AA). Plasma levels of IL-6 correlated inverselywith diuretic efficiency only in the patients having at least one copyof the major allele of the TMPRSS6 rs855791 SNP; plasma levels of IL-6were not significantly correlated with diuretic efficiency in patientshomozygous for the minor allele.

These data suggested that diuretic resistance (low diuretic efficiency)in heart failure patients could be treated with an IL-6 antagonist, butonly in those having at least one copy of the TMPRSS6 rs855791 majorallele.

In mouse M1 CCD cells, which are genotypically analogous to human cellshomozygous for the TMPRSS6 rs855791 major allele, the addition of IL-6correlated with the expression of ion transporters, NKCC2, ENaC-beta,and NCC. Increased expression of these ion transporters provides aputative mechanism for IL-6 mediated diuretic resistance.

Because the IL-6 mediated increase in expression of ion transporters isnot known to be linked to hepcidin expression, these data suggested thatIL-6 antagonism could also be effective in treating diuretic resistancein patients homozygous for the TMPRSS6 rs855791 minor allele.

Secondary analysis of data from two additional large heart failureclinical trials confirmed the association of diuretic resistance withIL-6 level (Example 5), independently of TMPRSS6 rs855791 genotype(Example 6), providing evidence that IL-6 antagonism should also beeffective in treating diuretic resistance in patients homozygous for theTMPRSS6 rs855791 minor allele.

Accordingly, in a first aspect, methods are provided for treating apatient who requires diuresis but is resistant to diuretics. The methodscomprise administering, in combination with a diuretic, atherapeutically effective amount of an IL-6 antagonist to the patient.

In some embodiments, the patient has elevated pre-treatment plasma IL-6levels. In certain embodiments, the patient has a pre-treatment plasmaIL-6 level of greater than 2 pg/mL. In certain embodiments, the patienthas a pre-treatment IL-6 level of greater than 3 pg/mL. In certainembodiments, the patient has a pre-treatment IL-6 level of greater than5 pg/mL. In certain embodiments, the patient has a pre-treatment IL-6level of greater than 10 pg/mL.

In some embodiments, the patient has a diuretic efficiency of less than500. In some embodiments, the patient has a diuretic efficiency of lessthan 200. In some embodiments, the patient has a diuretic efficiency ofless than 150. In some embodiments, the patient has a diureticefficiency of less than 100.

In some embodiments, the patient has diuretic resistant heart failure.In certain embodiments, the patient has acute heart failure. In certainembodiments, the patient has chronic heart failure.

In some embodiments, the patient has cardiorenal syndrome. In some ofthese embodiments, the patient has cardiorenal syndrome type 4.

In some embodiments, the patient has kidney disease. In certainembodiments, the patient has hepatorenal syndrome.

In some embodiments, the patient has at least one copy of the TMPRSS6rs855791 major allele.

In certain embodiments, the IL-6 antagonist is an anti-IL-6 antibody, orantigen-binding fragment or derivative thereof. In particularembodiments, the anti-IL-6 antibody or antigen-binding fragment orderivative has a K_(D) for binding human IL-6 of less than 100 nM, lessthan 50 nM, less than 10 nM, even less than 1 nM.

In certain embodiments, the anti-IL-6 antibody or antigen-bindingfragment or derivative has an elimination half-life followingintravenous administration of at least 7 days, at least 14 days, atleast 21 days, or at least 30 days.

In certain embodiments, the IL-6 antagonist is a full-length monoclonalanti-IL-6 antibody. In particular embodiments, the antibody is an IgG1or IgG4 antibody. In certain embodiments, the antibody is an IgG1antibody.

In certain embodiments, the anti-IL-6 antibody or antigen-bindingfragment or derivative is fully human. In certain embodiments, theanti-IL-6 antibody or antigen-binding fragment or derivative ishumanized.

In certain embodiments, the anti-IL-6 antibody or antigen-bindingfragment or derivative comprises all six variable region CDRs ofMED5117. In specific embodiments, the antibody comprises the VH and VLof MED5117. In particular embodiments, the antibody is MED5117.

In certain embodiments, the anti-IL-6 antibody or antigen-bindingfragment or derivative comprises all six variable region CDRs of anantibody selected from the group consisting of siltuximab, gerilimzumab,sirukumab, clazakizumab, olokizumab, elsilimomab, VX30 (VOP-R003;Vaccinex), EB-007 (EBI-029; Eleven Bio), ARGX-109 (ArGEN-X), FM101(Femta Pharmaceuticals, Lonza) and ALD518/BMS-945429 (AlderBiopharmaceuticals, Bristol-Myers Squibb). In certain embodiments, theanti-IL-6 antibody or antigen-binding fragment or derivative comprisesthe heavy chain V region and light chain V region from an antibodyselected from the group consisting of siltuximab, gerilimzumab,sirukumab, clazakizumab, olokizumab, VX30 (VOP-R003; Vaccinex), EB-007(EBI-029; Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals,Lonza) and ALD518/BMS-945429 (Alder Biopharmaceuticals, Bristol-MyersSquibb). In particular embodiments, the anti-IL-6 antibody orantigen-binding fragment or derivative is an antibody selected from thegroup consisting of siltuximab, gerilimzumab, sirukumab, clazakizumab,olokizumab, VX30 (VOP-R003; Vaccinex), EB-007 (EBI-029; Eleven Bio),ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza) andALD518/BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb).

In certain embodiments, the IL-6 antagonist is a single domain antibody,a Vim Nanobody, an Fab, or a scFv.

In certain embodiments, the IL-6 antagonist is an anti-IL-6R antibody,or antigen-binding fragment or derivative thereof. In certainembodiments, the anti-IL-6R antibody, antigen-binding fragment, orderivative comprises the 6 CDRs of tocilizumab or vobarilizumab.

In certain embodiments, the IL-6 antagonist is a JAK inhibitor. Incertain embodiments, the JAK inhibitor is selected from the groupconsisting of tofacitinib (Xeljanz), decemotinib, ruxolitinib,upadacitinib, baricitinib, filgotinib, lestaurtinib, pacritinib,peficitinib, INCB-039110, ABT-494, INCB-047986 and AC-410.

In certain embodiments, the IL-6 antagonist is a STAT3 inhibitor.

In certain embodiments, the IL-6 antagonist is administeredparenterally. In particular embodiments, the IL-6 antagonist isadministered subcutaneously.

In certain embodiments, the IL-6 antagonist is administered orally.

In certain embodiments, the IL-6 antagonist is administered at a dose,on a schedule, and for a period sufficient to increase diureticefficiency. In certain embodiments, the IL-6 antagonist is administeredat a dose, on a schedule, and for a period sufficient to increasediuretic efficiency to normal levels. In certain embodiments, the IL-6antagonist is administered at a dose, on a schedule, and for a periodsufficient to increase eGFR. In particular embodiments, the IL-6antagonist is administered at a dose, on a schedule, and for a periodsufficient to increase eGFR to normal levels.

In certain embodiments, the method further comprises the subsequent stepof determining the level of IL-6 in urine, determining the level of IL-6in plasma, or determining the level of IL-6 in urine and in plasma. Inparticular embodiments, the method further comprises a final step ofadjusting the dose of IL-6 antagonist for subsequent administrationbased on IL-6 level determined in the immediately preceding step.

7. BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1A is a bar graph showing diuretic efficiency (“DE”) by tertiles ofurine IL-6 in 129 heart failure (“HF”) patients receiving high dosediuretic therapy who were enrolled in the study described in Example 1herein. Whiskers extend from the 10^(th) to 90^(th) percentile.

FIG. 1B is a bar graph showing diuretic efficiency (“DE”) by tertiles ofplasma IL-6 in the 129 heart failure (“HF”) patients whose data is shownin FIG. 1A. Whiskers extend from the 10th to 90th percentile.

FIG. 2A plots DE by tertiles of urine IL-6 in the patients reported inFIGS. 1A and 1B further stratified by genotype, with the left panelshowing results for patients homozygous for the TMPRSS6 rs855791 SNPminor allele (2321G→A; A736V) and right panel showing results forpatients having at least one copy of the TMPRSS6 rs855791 SNP majorallele (2321G; A736).

FIG. 2B plots DE by tertiles of plasma IL-6 in the patients reported inFIGS. 1A and 1B further stratified by genotype, with the left panelshowing results for patients homozygous for the TMPRSS6 rs855791 SNPminor allele (2321G→A; A736V) and right panel showing results forpatients having at least one copy of the TMPRSS6 rs855791 SNP majorallele (2321G; A736).

FIGS. 3A and 3B plot the association in a 98 patient subset of the 129patients reported in FIGS. 1 and 2 of (A) Urine IL-6 levels and (B)Plasma IL-6 levels with the following clinical determinations: Reducedkidney function, low diuretic efficiency (“DE”), increased neurohormonalactivation, and risk of mortality. Whiskers represent 95% confidenceinterval (“CI”). All analyses adjusted for both urine and plasma levelsof IL-6. Urine IL-6 levels are indexed to urinary creatinine. Due to theskewed distribution of urine and plasma IL-6 variables, a log transformwas applied before performing logistic and Cox regressions.Abbreviations: OR=Odds ratio. HR=Hazards ratio. IL=interleukin.SD=standard deviation. eGFR=estimated glomerular filtration rate.*=adjusted for use of angiotensin converting enzyme inhibitor (ACE-I) orangiotensin receptor blocker (ARB). **=adjusted for baselinecharacteristics including age, race, amino terminal pro B-typenatriuretic peptide (NT-proBNP), use of ACE-I or ARB, home loop diureticdose, and eGFR.

FIGS. 4A, 4B, and 4C show the expression of NKCC2, ENaC-beta, and NCC inM1 CCD cells after treatment with IL-6 and/or Ruxolitinib, with FIG. 4Ashowing the expression of NKCC2 after the treatment with IL-6 and/orRuxolitinib; FIG. 4B showing the expression of ENaC-beta after thetreatment with IL-6 and/or Ruxolitinib; and FIG. 4C showing theexpression of NCC after the treatment with IL-6 and/or Ruxolitinib.

FIGS. 5A, 5B, and 5C show the expression of NKCC2, ENaC-beta, and NCC inM1 CCD cells after treatment with IL-6 and/or Spironolactone, with FIG.5A showing the expression of NKCC2 after the treatment with IL-6 and/orSpironolactone; FIG. 5B showing the expression of ENaC-beta after thetreatment with IL-6 and/or Spironolactone; and FIG. 5C showing theexpression of NCC after the treatment with IL-6 and/or Spironolactone.

FIG. 6 shows the association of baseline characteristics with higherlevels of IL-6 in the PROTECT trial described in Example 5.

FIGS. 7A and 7B show the Kaplan-Meier survival curve by tertiles ofIL-6, with FIG. 7A showing the all-cause mortality at 180 days and FIG.7B showing all-cause mortality or cardiovascular relatedrehospitalization at 60 days.

FIGS. 8A and 8B show the Kaplan-Meier survival curve for change of IL-6between baseline and day 7, with FIG. 8A showing the all-cause mortalityat 180 days and FIG. 8B showing all-cause mortality or cardiovascularrelated rehospitalization at 60 days.

FIG. 9 shows the association of baseline characteristics with higherlevels of IL-6 in the BIOSTAT-CHF study described in Example 6.

FIGS. 10A and 10B show the Kaplan-Meier survival curve by tertiles ofIL-6, with FIG. 10A showing the all-cause mortality and/orrehospitalization for heart failure at 2 years and FIG. 10B showingall-cause mortality at 2 years.

FIG. 11 show the analysis between ferritin levels and tertiles of IL-6.

8. DETAILED DESCRIPTION 8.1. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting.

By “transmembrane protease serine 6 (TMPRSS6) polypeptide” is meant apolypeptide or fragment thereof having at least about 85% or greateramino acid identity to the amino acid sequence provided at NCBIAccession No. NP_001275929 and having serine proteinase activity. TheTMPRSS6 polypeptide, also known as Matriptase-2 (MT2), cleaveshemojuvelin and inhibits bone morphogenetic protein signaling. Anexemplary TMPRSS6 amino acid sequence having an alanine at position 736(736A) is provided below:

(SEQ ID NO: 1)  1 MPVAEAPQVA GGQGDGGDGE EAEPEGMFKA CEDSKRKARG YLRLVPLFVL LALLVLASAG  61 VLLWYFLGYK AEVMVSQVYS GSLRVLNRHF SQDLTRRESS AFRSETAKAQ KMLKELITST 121 RLGTYYNSSS VYSFGEGPLT CFFWFILQIP EHRRLMLSPE VVQALLVEEL LSTVNSSAAV 181 PYRAEYEVDP EGLVILEASV KDIAALNSTL GCYRYSYVGQ GQVLRLKGPD HLASSCLWHL 241 QGPKDLMLKL RLEWTLAECR DRLAMYDVAG PLEKRLITSV YGCSRQEPVV EVLASGAIMA 301 VVWKKGLHSY YDPFVLSVQP VVFQACEVNL TLDNRLDSQG VLSTPYFPSY YSPQTHCSWH 361 LTVPSLDYGL ALWFDAYALR RQKYDLPCTQ GQWTIQNRRL CGLRILQPYA ERIPVVATAG 421 ITINFTSQIS LTGPGVRVHY GLYNQSDPCP GEFLCSVNGL CVPACDGVKD CPNGLDERNC 481 VCRATFQCKE DSTCISLPKV CDGQPDCLNG SDEEQCQEGV PCGTFTFQCE DRSCVKKPNP 541 QCDGRPDCRD GSDEEHCDCG LQGPSSRIVG GAVSSEGEWP WQASLQVRGR HICGGALIAD 601 RWVITAAHCF QEDSMASTVL WTVFLGKVWQ NSRWPGEVSF KVSRLLLHPY HEEDSHDYDV 661 ALLQLDHPVV RSAAVRPVCL PARSHFFEPG LHCWITGWGA LREGALRADA VALFYGWRNQ 721 GSETCCCPIS NALQKADVQL IPQDLCSEVY RYQVTPRMLC AGYRKGKKDA CQGDSGGPLV 781 CKALSGRWFL AGLVSWGLGC GRPNYFGVYT RITGVISWIQ QVVT 

An exemplary TMPRSS6 amino acid sequence having a valine at position 736(736V) is provided below:

(SEQ ID NO: 2)  1 MPVAEAPQVA GGQGDGGDGE EAEPEGMFKA CEDSKRKARG YLRLVPLFVL LALLVLASAG  61 VLLWYFLGYK AEVMVSQVYS GSLRVLNRHF SQDLTRRESS AFRSETAKAQ KMLKELITST 121 RLGTYYNSSS VYSFGEGPLT CFFWFILQIP EHRRLMLSPE VVQALLVEEL LSTVNSSAAV 181 PYRAEYEVDP EGLVILEASV KDIAALNSTL GCYRYSYVGQ GQVLRLKGPD HLASSCLWHL  24 QGPKDLMLKL RLEWTLAECR DRLAMYDVAG PLEKRLITSV YGCSRQEPVV EVLASGAIMA  30 VVWKKGLHSY YDPFVLSVQP VVFQACEVNL TLDNRLDSQG VLSTPYFPSY YSPQTHCSWH  36 LTVPSLDYGL ALWFDAYALR RQKYDLPCTQ GQWTIQNRRL CGLRILQPYA ERIPVVATAG  42 ITINFTSQIS LTGPGVRVHY GLYNQSDPCP GEFLCSVNGL CVPACDGVKD CPNGLDERNC  48 VCRATFQCKE DSTCISLPKV CDGQPDCLNG SDEEQCQEGV PCGTFTFQCE DRSCVKKPNP  54 QCDGRPDCRD GSDEEHCDCG LQGPSSRIVG GAVSSEGEWP WQASLQVRGR HICGGALIAD  60 RWVITAAHCF QEDSMASTVL WTVFLGKVWQ NSRWPGEVSF KVSRLLLHPY HEEDSHDYDV  66 ALLQLDHPVV RSAAVRPVCL PARSHFFEPG LHCWITGWGA LREGALRADA VALFYGWRNQ  72 GSETCCCPIS NALQKVDVQL IPQDLCSEVY RYQVTPRMLC AGYRKGKKDA CQGDSGGPLV  78 CKALSGRWFL AGLVSWGLGC GRPNYFGVYT RITGVISWIQ QVVT 

By “TMPRSS6 nucleic acid molecule” is meant a polynucleotide encoding aTMPRSS6 polypeptide (Matriptase-2; MT2). An exemplary TMPRSS6 nucleicacid molecule sequence is provided at NCBI Accession No. NM_001289000. ATMPRSS6 nucleic acid sequence having a G at nucleotide position 2321 (“Gallele”; “major allele”) is provided below:

(SEQ ID NO: 3)   1 GGACAAACAG AGGCTCCTGA GGCCTGTGTG CAGGCCCGGC ACCTATCTGC CGCTCCCAAA  61 GGATGCCCGT GGCCGAGGCC CCCCAGGTGG CTGGCGGGCA GGGGGACGGA GGTGATGGCG 121 AGGAAGCGGA GCCGGAGGGG ATGTTCAAGG CCTGTGAGGA CTCCAAGAGA AAAGCCCGGG 181 GCTACCTCCG CCTGGTGCCC CTGTTTGTGC TGCTGGCCCT GCTCGTGCTG GCTTCGGCGG 241 GGGTGCTACT CTGGTATTTC CTAGGGTACA AGGCGGAGGT GATGGTCAGC CAGGTGTACT 301 CAGGCAGTCT GCGTGTACTC AATCGCCACT TCTCCCAGGA TCTTACCCGC CGGGAATCTA 361 GTGCCTTCCG CAGTGAAACC GCCAAAGCCC AGAAGATGCT CAAGGAGCTC ATCACCAGCA 421 CCCGCCTGGG AACTTACTAC AACTCCAGCT CCGTCTATTC CTTTGGGGAG GGACCCCTCA 481 CCTGCTTCTT CTGGTTCATT CTCCAAATCC CCGAGCACCG CCGGCTGATG CTGAGCCCCG 541 AGGTGGTGCA GGCACTGCTG GTGGAGGAGC TGCTGTCCAC AGTCAACAGC TCGGCTGCCG 601 TCCCCTACAG GGCCGAGTAC GAAGTGGACC CCGAGGGCCT AGTGATCCTG GAAGCCAGTG 661 TGAAAGACAT AGCTGCATTG AATTCCACGC TGGGTTGTTA CCGCTACAGC TACGTGGGCC 721 AGGGCCAGGT CCTCCGGCTG AAGGGGCCTG ACCACCTGGC CTCCAGCTGC CTGTGGCACC 781 TGCAGGGCCC CAAGGACCTC ATGCTCAAAC TCCGGCTGGA GTGGACGCTG GCAGAGTGCC 841 GGGACCGACT GGCCATGTAT GACGTGGCCG GGCCCCTGGA GAAGAGGCTC ATCACCTCGG 901 TGTACGGCTG CAGCCGCCAG GAGCCCGTGG TGGAGGTTCT GGCGTCGGGG GCCATCATGG 961 CGGTCGTCTG GAAGAAGGGC CTGCACAGCT ACTACGACCC CTTCGTGCTC TCCGTGCAGC1021 CGGTGGTCTT CCAGGCCTGT GAAGTGAACC TGACGCTGGA CAACAGGCTC GACTCCCAGG1081 GCGTCCTCAG CACCCCGTAC TTCCCCAGCT ACTACTCGCC CCAAACCCAC TGCTCCTGGC1141 ACCTCACGGT GCCCTCTCTG GACTACGGCT TGGCCCTCTG GTTTGATGCC TATGCACTGA1201 GGAGGCAGAA GTATGATTTG CCGTGCACCC AGGGCCAGTG GACGATCCAG AACAGGAGGC1261 TGTGTGGCTT GCGCATCCTG CAGCCCTACG CCGAGAGGAT CCCCGTGGTG GCCACGGCCG1321 GGATCACCAT CAACTTCACC TCCCAGATCT CCCTCACCGG GCCCGGTGTG CGGGTGCACT1381 ATGGCTTGTA CAACCAGTCG GACCCCTGCC CTGGAGAGTT CCTCTGTTCT GTGAATGGAC1441 TCTGTGTCCC TGCCTGTGAT GGGGTCAAGG ACTGCCCCAA CGGCCTGGAT GAGAGAAACT1501 GCGTTTGCAG AGCCACATTC CAGTGCAAAG AGGACAGCAC ATGCATCTCA CTGCCCAAGG1561 TCTGTGATGG GCAGCCTGAT TGTCTCAACG GCAGCGACGA AGAGCAGTGC CAGGAAGGGG1621 TGCCATGTGG GACATTCACC TTCCAGTGTG AGGACCGGAG CTGCGTGAAG AAGCCCAACC1681 CGCAGTGTGA TGGGCGGCCC GACTGCAGGG ACGGCTCGGA TGAGGAGCAC TGTGACTGTG1741 GCCTCCAGGG CCCCTCCAGC CGCATTGTTG GTGGAGCTGT GTCCTCCGAG GGTGAGTGGC1801 CATGGCAGGC CAGCCTCCAG GTTCGGGGTC GACACATCTG TGGGGGGGCC CTCATCGCTG1861 ACCGCTGGGT GATAACAGCT GCCCACTGCT TCCAGGAGGA CAGCATGGCC TCCACGGTGC1921 TGTGGACCGT GTTCCTGGGC AAGGTGTGGC AGAACTCGCG CTGGCCTGGA GAGGTGTCCT1981 TCAAGGTGAG CCGCCTGCTC CTGCACCCGT ACCACGAAGA GGACAGCCAT GACTACGACG2041 TGGCGCTGCT GCAGCTCGAC CACCCGGTGG TGCGCTCGGC CGCCGTGCGC CCCGTCTGCC2101 TGCCCGCGCG CTCCCACTTC TTCGAGCCCG GCCTGCACTG CTGGATTACG GGCTGGGGCG2161 CCTTGCGCGA GGGCGCCCTA CGGGCGGATG CTGTGGCCCT ATTTTATGGA TGGAGAAACC2221 AAGGCTCAGA GACATGTTGC TGCCCCATCA GCAACGCTCT GCAGAAAGTG GATGTGCAGT2281 TGATCCCACA GGACCTGTGC AGCGAGGTCT ATCGCTACCA GGTGACGCCA CGCATGCTGT2341 GTGCCGGCTA CCGCAAGGGC AAGAAGGATG CCTGTCAGGG TGACTCAGGT GGTCCGCTGG2401 TGTGCAAGGC ACTCAGTGGC CGCTGGTTCC TGGCGGGGCT GGTCAGCTGG GGCCTGGGCT2461 GTGGCCGGCC TAACTACTTC GGCGTCTACA CCCGCATCAC AGGTGTGATC AGCTGGATCC2521 AGCAAGTGGT GACCTGAGGA ACTGCCCCCC TGCAAAGCAG GGCCCACCTC CTGGACTCAG2581 AGAGCCCAGG GCAACTGCCA AGCAGGGGGA CAAGTATTCT GGCGGGGGGT GGGGGAGAGA2641 GCAGGCCCTG TGGTGGCAGG AGGTGGCATC TTGTCTCGTC CCTGATGTCT GCTCCAGTGA2701 TGGCAGGAGG ATGGAGAAGT GCCAGCAGCT GGGGGTCAAG ACGTCCCCTG AGGACCCAGG2761 CCCACACCCA GCCCTTCTGC CTCCCAATTC TCTCTCCTCC GTCCCCTTCC TCCACTGCTG2821 CCTAATGCAA GGCAGTGGCT CAGCAGCAAG AATGCTGGTT CTACATCCCG AGGAGTGTCT2881 GAGGTGCGCC CCACTCTGTA CAGAGGCTGT TTGGGCAGCC TTGCCTCCAG AGAGCAGATT2941 CCAGCTTCGG AAGCCCCTGG TCTAACTTGG GATCTGGGAA TGGAAGGTGC TCCCATCGGA3001 GGGGACCCTC AGAGCCCTGG AGACTGCCAG GTGGGCCTGC TGCCACTGTA AGCCAAAAGG3061 TGGGGAAGTC CTGACTCCAG GGTCCTTGCC CCACCCCTGC CTGCCACCTG GGCCCTCACA3121 GCCCAGACCC TCACTGGGAG GTGAGCTCAG CTGCCCTTTG GAATAAAGCT GCCTGATCCA3181 AAAAAAAAAA AAAAAA 

A TMPRSS6 nucleic acid sequence having an A at nucleotide position 2321is provided below:

(SEQ ID NO: 4)   1 GGACAAACAG AGGCTCCTGA GGCCTGTGTG CAGGCCCGGC ACCTATCTGC CGCTCCCAAA   61 GGATGCCCGT GGCCGAGGCC CCCCAGGTGG CTGGCGGGCA GGGGGACGGA GGTGATGGCG  121 AGGAAGCGGA GCCGGAGGGG ATGTTCAAGG CCTGTGAGGA CTCCAAGAGA AAAGCCCGGG  181 GCTACCTCCG CCTGGTGCCC CTGTTTGTGC TGCTGGCCCT GCTCGTGCTG GCTTCGGCGG  241 GGGTGCTACT CTGGTATTTC CTAGGGTACA AGGCGGAGGT GATGGTCAGC CAGGTGTACT  301 CAGGCAGTCT GCGTGTACTC AATCGCCACT TCTCCCAGGA TCTTACCCGC CGGGAATCTA  361 GTGCCTTCCG CAGTGAAACC GCCAAAGCCC AGAAGATGCT CAAGGAGCTC ATCACCAGCA  421 CCCGCCTGGG AACTTACTAC AACTCCAGCT CCGTCTATTC CTTTGGGGAG GGACCCCTCA  481 CCTGCTTCTT CTGGTTCATT CTCCAAATCC CCGAGCACCG CCGGCTGATG CTGAGCCCCG  541 AGGTGGTGCA GGCACTGCTG GTGGAGGAGC TGCTGTCCAC AGTCAACAGC TCGGCTGCCG  601 TCCCCTACAG GGCCGAGTAC GAAGTGGACC CCGAGGGCCT AGTGATCCTG GAAGCCAGTG  661 TGAAAGACAT AGCTGCATTG AATTCCACGC TGGGTTGTTA CCGCTACAGC TACGTGGGCC  721 AGGGCCAGGT CCTCCGGCTG AAGGGGCCTG ACCACCTGGC CTCCAGCTGC CTGTGGCACC  781 TGCAGGGCCC CAAGGACCTC ATGCTCAAAC TCCGGCTGGA GTGGACGCTG GCAGAGTGCC  841 GGGACCGACT GGCCATGTAT GACGTGGCCG GGCCCCTGGA GAAGAGGCTC ATCACCTCGG  901 TGTACGGCTG CAGCCGCCAG GAGCCCGTGG TGGAGGTTCT GGCGTCGGGG GCCATCATGG  961 CGGTCGTCTG GAAGAAGGGC CTGCACAGCT ACTACGACCC CTTCGTGCTC TCCGTGCAGC 1021 CGGTGGTCTT CCAGGCCTGT GAAGTGAACC TGACGCTGGA CAACAGGCTC GACTCCCAGG 1081 GCGTCCTCAG CACCCCGTAC TTCCCCAGCT ACTACTCGCC CCAAACCCAC TGCTCCTGGC 1141 ACCTCACGGT GCCCTCTCTG GACTACGGCT TGGCCCTCTG GTTTGATGCC TATGCACTGA 1201 GGAGGCAGAA GTATGATTTG CCGTGCACCC AGGGCCAGTG GACGATCCAG AACAGGAGGC 1261 TGTGTGGCTT GCGCATCCTG CAGCCCTACG CCGAGAGGAT CCCCGTGGTG GCCACGGCCG 1321 GGATCACCAT CAACTTCACC TCCCAGATCT CCCTCACCGG GCCCGGTGTG CGGGTGCACT 1381 ATGGCTTGTA CAACCAGTCG GACCCCTGCC CTGGAGAGTT CCTCTGTTCT GTGAATGGAC 1441 TCTGTGTCCC TGCCTGTGAT GGGGTCAAGG ACTGCCCCAA CGGCCTGGAT GAGAGAAACT 1501 GCGTTTGCAG AGCCACATTC CAGTGCAAAG AGGACAGCAC ATGCATCTCA CTGCCCAAGG 1561 TCTGTGATGG GCAGCCTGAT TGTCTCAACG GCAGCGACGA AGAGCAGTGC CAGGAAGGGG 1621 TGCCATGTGG GACATTCACC TTCCAGTGTG AGGACCGGAG CTGCGTGAAG AAGCCCAACC 1681 CGCAGTGTGA TGGGCGGCCC GACTGCAGGG ACGGCTCGGA TGAGGAGCAC TGTGACTGTG 1741 GCCTCCAGGG CCCCTCCAGC CGCATTGTTG GTGGAGCTGT GTCCTCCGAG GGTGAGTGGC 1801 CATGGCAGGC CAGCCTCCAG GTTCGGGGTC GACACATCTG TGGGGGGGCC CTCATCGCTG 1861 ACCGCTGGGT GATAACAGCT GCCCACTGCT TCCAGGAGGA CAGCATGGCC TCCACGGTGC 1921 TGTGGACCGT GTTCCTGGGC AAGGTGTGGC AGAACTCGCG CTGGCCTGGA GAGGTGTCCT 1981 TCAAGGTGAG CCGCCTGCTC CTGCACCCGT ACCACGAAGA GGACAGCCAT GACTACGACG 2041 TGGCGCTGCT GCAGCTCGAC CACCCGGTGG TGCGCTCGGC CGCCGTGCGC CCCGTCTGCC 2101 TGCCCGCGCG CTCCCACTTC TTCGAGCCCG GCCTGCACTG CTGGATTACG GGCTGGGGCG 2161 CCTTGCGCGA GGGCGCCCTA CGGGCGGATG CTGTGGCCCT ATTTTATGGA TGGAGAAACC 2221 AAGGCTCAGA GACATGTTGC TGCCCCATCA GCAACGCTCT GCAGAAAGTG GATGTGCAGT 2281 TGATCCCACA GGACCTGTGC AGCGAGGTCT ATCGCTACCA AGTGACGCCA CGCATGCTGT 2341 GTGCCGGCTA CCGCAAGGGC AAGAAGGATG CCTGTCAGGG TGACTCAGGT GGTCCGCTGG 2401 TGTGCAAGGC ACTCAGTGGC CGCTGGTTCC TGGCGGGGCT GGTCAGCTGG GGCCTGGGCT 2461 GTGGCCGGCC TAACTACTTC GGCGTCTACA CCCGCATCAC AGGTGTGATC AGCTGGATCC 2521 AGCAAGTGGT GACCTGAGGA ACTGCCCCCC TGCAAAGCAG GGCCCACCTC CTGGACTCAG 2581 AGAGCCCAGG GCAACTGCCA AGCAGGGGGA CAAGTATTCT GGCGGGGGGT GGGGGAGAGA 2641 GCAGGCCCTG TGGTGGCAGG AGGTGGCATC TTGTCTCGTC CCTGATGTCT GCTCCAGTGA 2701 TGGCAGGAGG ATGGAGAAGT GCCAGCAGCT GGGGGTCAAG ACGTCCCCTG AGGACCCAGG 2761 CCCACACCCA GCCCTTCTGC CTCCCAATTC TCTCTCCTCC GTCCCCTTCC TCCACTGCTG 2821 CCTAATGCAA GGCAGTGGCT CAGCAGCAAG AATGCTGGTT CTACATCCCG AGGAGTGTCT 2881 GAGGTGCGCC CCACTCTGTA CAGAGGCTGT TTGGGCAGCC TTGCCTCCAG AGAGCAGATT 2941 CCAGCTTCGG AAGCCCCTGG TCTAACTTGG GATCTGGGAA TGGAAGGTGC TCCCATCGGA 3001 GGGGACCCTC AGAGCCCTGG AGACTGCCAG GTGGGCCTGC TGCCACTGTA AGCCAAAAGG 3061 TGGGGAAGTC CTGACTCCAG GGTCCTTGCC CCACCCCTGC CTGCCACCTG GGCCCTCACA 3121 GCCCAGACCC TCACTGGGAG GTGAGCTCAG CTGCCCTTTG GAATAAAGCT GCCTGATCCA 3181 AAAAAAAAAA AAAAAA 

By “variant” is meant a polynucleotide or polypeptide sequence thatdiffers from a reference sequence by one or more nucleotides or one ormore amino acids. An exemplary TMPRSS6 variant is TMPRSS6 (A736V),resulting from SNP rs855791 (G→A).

By “single nucleotide polymorphism” or “SNP” is meant a naturallyoccurring DNA sequence variant in which a single nucleotide in thegenome differs between members of a biological species or between pairedchromosomes in an individual. SNPs can be used as genetic markers forvariant alleles. In one embodiment, the TMPRSS6 SNP is rs855791.

By “rs855791” is meant a single nucleotide polymorphism (SNP) in thehuman TMPRSS6 gene, 2321G→A, resulting in an alanine to valinesubstitution (A736V) in the catalytic domain of Matriptase-2 (MT2),which is encoded by the TMPRSS6 gene. The allele with highest frequencyin the human population (the major allele) is 2321G, encoding 736A. Theallele with lowest frequency in the human population (minor allele) is2321A, encoding 736V.

By “heterozygous” is meant that a chromosomal locus has two differentalleles. In one embodiment of the methods described herein, heterozygousrefers to a genotype in which one allele has a TMPRSS6 nucleic acidsequence encoding a TMPRSS6 polypeptide having an alanine at amino acidposition 736 (e.g., having a G or C at nucleotide position 2321 of aTMPRSS6 nucleic acid molecule) (rs855791 major allele), and the otherallele has a variant TMPRSS6 nucleic acid sequence encoding a TMPRSS6polypeptide comprising a valine at amino acid position 736 (e.g., havingan A or T at nucleotide position 2321 of a TMPRSS6 nucleic acidmolecule) (rs855791 minor allele).

By “homozygous” is meant that a chromosomal locus has two identicalalleles. In certain embodiments of the methods described herein,homozygous refers to a genotype in which both alleles have a TMPRSS6nucleic acid sequence encoding a TMPRSS6 polypeptide comprising analanine at amino acid position 736 (e.g., having a G or C at nucleotideposition 2321 of a TMPRSS6 nucleic acid molecule) (rs855791 homozygousmajor allele). In certain embodiments, homozygous refers to a genotypein which both alleles have a TMPRSS6 nucleic acid sequence encoding aTMPRSS6 polypeptide comprising a valine at amino acid position 736(e.g., having an A or T at nucleotide position 2321 of a TMPRSS6 nucleicacid molecule) (rs855791 homozygous minor allele).

“Determining that a patient has at least one copy of the TMPRSS6rs855791 major allele” includes, but is not limited to, performing anassay to determine that a patient has at least one copy of the TMPRSS6rs855791 major allele; ordering an assay to determine that a patient hasat least one copy of the TMPRSS6 rs855791 major allele; prescribing anassay to determine that a patient has at least one copy of the TMPRSS6rs855791 major allele; otherwise directing or controlling that an assaybe performed to determine that a patient has at least one copy of theTMPRSS6 rs855791 major allele; and reviewing TMRSS6 genotype assay dataor protein or nucleic acid sequence data to determine that a patient hasat least one copy of the TMPRSS6 rs855791 major allele.

By “interleukin 6” or “IL-6” or “IL-6 polypeptide” is meant apolypeptide or fragment thereof having at least about 85% or greateramino acid identity to the amino acid sequence provided at NCBIAccession No. NP_000591 and having IL-6 biological activity. IL-6 is apleotropic cytokine with multiple biologic functions. Exemplary IL-6biological activities include immunostimulatory and pro-inflammatoryactivities. An exemplary IL-6 amino acid sequence is provided below:

(SEQ ID NO: 5)  1 MCVGARRLGR GPCAALLLLG LGLSTVTGLH CVGDTYPSND RCCHECRPGN GMVSRCSRSQ 61 NTVCRPCGPG FYNDVVSSKP CKPCTWCNLR SGSERKQLCT ATQDTVCRCR AGTQPLDSYK121 PGVDCAPCPP GHFSPGDNQA CKPWTNCTLA GKHTLQPASN SSDAICEDRD PPATQPQETQ181 GPPARPITVQ PTEAWPRTSQ GPSTRPVEVP GGRAVAAILG LGLVLGLLGP LAILLALYLL241 RRDQRLPPDA HKPPGGGSFR TPIQEEQADA HSTLAKI 

By “interleukin 6 (IL-6) nucleic acid” is meant a polynucleotideencoding an interleukin 6 (IL-6) polypeptide. An exemplary interleukin 6(IL-6) nucleic acid sequence is provided at NCBI Accession No. NM000600. The exemplary sequence at NCBI Accession No. NM_000600 isprovided below.

(SEQ ID NO: 6)   1 AATATTAGAG TCTCAACCCC CAATAAATAT AGGACTGGAG ATGTCTGAGG CTCATTCTGC   61 CCTCGAGCCC ACCGGGAACG AAAGAGAAGC TCTATCTCCC CTCCAGGAGC CCAGCTATGA  121 ACTCCTTCTC CACAAGCGCC TTCGGTCCAG TTGCCTTCTC CCTGGGGCTG CTCCTGGTGT  181 TGCCTGCTGC CTTCCCTGCC CCAGTACCCC CAGGAGAAGA TTCCAAAGAT GTAGCCGCCC  241 CACACAGACA GCCACTCACC TCTTCAGAAC GAATTGACAA ACAAATTCGG TACATCCTCG  301 ACGGCATCTC AGCCCTGAGA AAGGAGACAT GTAACAAGAG TAACATGTGT GAAAGCAGCA  361 AAGAGGCACT GGCAGAAAAC AACCTGAACC TTCCAAAGAT GGCTGAAAAA GATGGATGCT  421 TCCAATCTGG ATTCAATGAG GAGACTTGCC TGGTGAAAAT CATCACTGGT CTTTTGGAGT  481 TTGAGGTATA CCTAGAGTAC CTCCAGAACA GATTTGAGAG TAGTGAGGAA CAAGCCAGAG  541 CTGTGCAGAT GAGTACAAAA GTCCTGATCC AGTTCCTGCA GAAAAAGGCA AAGAATCTAG  601 ATGCAATAAC CACCCCTGAC CCAACCACAA ATGCCAGCCT GCTGACGAAG CTGCAGGCAC  661 AGAACCAGTG GCTGCAGGAC ATGACAACTC ATCTCATTCT GCGCAGCTTT AAGGAGTTCC  721 TGCAGTCCAG CCTGAGGGCT CTTCGGCAAA TGTAGCATGG GCACCTCAGA TTGTTGTTGT  781 TAATGGGCAT TCCTTCTTCT GGTCAGAAAC CTGTCCACTG GGCACAGAAC TTATGTTGTT  841 CTCTATGGAG AACTAAAAGT ATGAGCGTTA GGACACTATT TTAATTATTT TTAATTTATT  901 AATATTTAAA TATGTGAAGC TGAGTTAATT TATGTAAGTC ATATTTATAT TTTTAAGAAG  961 TACCACTTGA AACATTTTAT GTATTAGTTT TGAAATAATA ATGGAAAGTG GCTATGCAGT 1021 TTGAATATCC TTTGTTTCAG AGCCAGATCA TTTCTTGGAA AGTGTAGGCT TACCTCAAAT 1081 AAATGGCTAA CTTATACATA TTTTTAAAGA AATATTTATA TTGTATTTAT ATAATGTATA 1141 AATGGTTTTT ATACCAATAA ATGGCATTTT AAAAAATTCA GCAAAAAAAA AAAAAAAAAA 1201 A 

By “interleukin 6 receptor (IL-6R) complex” is meant a protein complexcomprising an IL-6 receptor subunit alpha (IL-6Rα) and interleukin 6signal transducer Glycoprotein 130, also termed interleukin 6 receptorsubunit β (IL-6Rβ).

By “interleukin 6 receptor subunit α (IL-6Rα) polypeptide” is meant apolypeptide or fragment thereof having at least about 85% or greateramino acid identity to the amino acid sequence provided at NCBIAccession No. NP_000556 or NP_852004 and having IL-6 receptor biologicalactivity. Exemplary IL-6Rα biological activities include binding toIL-6, binding to glycoprotein 130 (gp130), and regulation of cell growthand differentiation. An exemplary IL-6R sequence is provided below:

(SEQ ID NO: 7)  1 MLAVGCALLA ALLAAPGAAL APRRCPAQEV ARGVLTSLPG DSVTLTCPGV EPEDNATVHW 61 VLRKPAAGSH PSRWAGMGRR LLLRSVQLHD SGNYSCYRAG RPAGTVHLLV DVPPEEPQLS121 CFRKSPLSNV VCEWGPRSTP SLTTKAVLLV RKFQNSPAED FQEPCQYSQE SQKFSCQLAV181 PEGDSSFYIV SMCVASSVGS KFSKTQTFQG CGILQPDPPA NITVTAVARN PRWLSVTWQD241 PHSWNSSFYR LRFELRYRAE RSKTFTTWMV KDLQHHCVIH DAWSGLRHVV QLRAQEEFGQ301 GEWSEWSPEA MGTPWTESRS PPAENEVSTP MQALTTNKDD DNILFRDSAN ATSLPVQDSS361 SVPLPTFLVA GGSLAFGTLL CIAIVLRFKK TWKLRALKEG KTSMHPPYSL GQLVPERPRP421 TPVLVPLISP PVSPSSLGSD NTSSHNRPDA RDPRSPYDIS NTDYFFPR 

By “interleukin 6 receptor subunit β (IL-6Rβ) polypeptide” is meant apolypeptide or fragment thereof having at least about 85% or greateramino acid identity to the amino acid sequence provided at NCBIAccession No. NP_002175, NP_786943, or NP_001177910 and having IL-6receptor biological activity. Exemplary IL-6Rβ biological activitiesinclude binding to IL-6Rα, IL-6 receptor signaling activity, andregulation of cell growth, differentiation, hepcidin expression etc. Anexemplary IL-6Rβ sequence is provided below:

(SEQ ID NO: 8)  1 MLTLQTWLVQ ALFIFLTTES TGELLDPCGY ISPESPVVQL HSNFTAVCVL KEKCMDYFHV 61 NANYIVWKTN HFTIPKEQYT IINRTASSVT FTDIASLNIQ LTCNILTFGQ LEQNVYGITI121 ISGLPPEKPK NLSCIVNEGK KMRCEWDGGR ETHLETNFTL KSEWATHKFA DCKAKRDTPT181 SCTVDYSTVY FVNIEVWVEA ENALGKVTSD HINFDPVYKV KPNPPHNLSV INSEELSSIL241 KLTWTNPSIK SVIILKYNIQ YRTKDASTWS QIPPEDTAST RSSFTVQDLK PFTEYVFRIR301 CMKEDGKGYW SDWSEEASGI TYEDRPSKAP SFWYKIDPSH TQGYRTVQLV WKTLPPFEAN361 GKILDYEVTL TRWKSHLQNY TVNATKLTVN LTNDRYLATL TVRNLVGKSD AAVLTIPACD421 FQATHPVMDL KAFPKDNMLW VEWTTPRESV KKYILEWCVL SDKAPCITDW QQEDGTVHRT481 YLRGNLAESK CYLITVTPVY ADGPGSPESI KAYLKQAPPS KGPTVRTKKV GKNEAVLEWD541 QLPVDVQNGF IRNYTIFYRT IIGNETAVNV DSSHTEYTLS SLTSDTLYMV RMAAYTDEGG601 KDGPEFTFTT PKFAQGEIEA IVVPVCLAFL LTTLLGVLFC FNKRDLIKKH IWPNVPDPSK661 SHIAQWSPHT PPRHNFNSKD QMYSDGNFTD VSVVEIEAND KKPFPEDLKS LDLFKKEKIN721 TEGHSSGIGG SSCMSSSRPS ISSSDENESS QNTSSTVQYS TVVHSGYRHQ VPSVQVFSRS781 ESTQPLLDSE ERPEDLQLVD HVDGGDGILP RQQYFKQNCS QHESSPDISH FERSKQVSSV841 NEEDFVRLKQ QISDHISQSC GSGQMKMFQE VSAADAFGPG TEGQVERFET VGMEAATDEG901 MPKSYLPQTV RQGGYMPQ 

By “IL-6 antagonist” is meant an agent that is capable of decreasing thebiological activity of IL-6. IL-6 antagonists include agents thatdecrease the level of IL-6 polypeptide in serum, including agents thatdecrease the expression of an IL-6 polypeptide or nucleic acid; agentsthat decrease the ability of IL-6 to bind to the IL-6R; agents thatdecrease the expression of the IL-6R; and agents that decrease signaltransduction by the IL-6R receptor when bound by IL-6. In preferredembodiments, the IL-6 antagonist decreases IL-6 biological activity byat least about 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%. Asfurther described in Section 6.3.4 below, IL-6 antagonists include IL-6binding polypeptides, such as anti-IL-6 antibodies and antigen bindingfragments or derivatives thereof; IL-6R binding polypeptides, such asanti-IL-6R antibodies and antigen binding fragments or derivativesthereof; and synthetic chemical molecules, such as JAK1 and JAK3inhibitors.

By “IL-6 antibody” or “anti-IL-6 antibody” is meant an antibody thatspecifically binds IL-6. Anti-IL-6 antibodies include monoclonal andpolyclonal antibodies that are specific for IL-6, and antigen-bindingfragments or derivatives thereof. IL-6 antibodies are described ingreater detail in Section 8.3.6.1 below.

As used herein, “diuretic efficiency” is calculated as mmol urinarysodium per doubling of loop diuretic dose (mmol Na/doubling of loopdiuretic dose) according to the methods described in Hanberg et al.,Circ. Heart Fail. 2016; 9:e003180, the disclosure of which isincorporated herein by reference in its entirety.

By “diuretic resistant heart failure” is meant heart failure in whichthe patient's diuretic efficiency is less than 100.

The terms “biomarker” or “marker,” as used herein, refers to a moleculethat can be detected. Therefore, a biomarker according to the presentinvention includes, but is not limited to, a nucleic acid, apolypeptide, a carbohydrate, a lipid, an inorganic molecule, an organicmolecule, each of which may vary widely in size and properties. A“biomarker” can be a bodily substance relating to a bodily condition ordisease. A “biomarker” can be detected using any means known in the artor by a previously unknown means that only becomes apparent uponconsideration of the marker by the skilled artisan.

As used herein, “biomarker” in the context of the present inventionencompasses, without limitation, proteins, nucleic acids, andmetabolites, together with their polymorphisms, mutations, variants,modifications, subunits, fragments, protein-ligand complexes, anddegradation products, elements, related metabolites, and other analytesor sample-derived measures. Biomarkers can also include mutated proteinsor mutated nucleic acids. Biomarkers also encompass non-blood bornefactors or non-analyte physiological markers of health status, such asclinical parameters, as well as traditional laboratory risk factors. Asdefined by the Food and Drug Administration (FDA), a biomarker is acharacteristic (e.g. measurable DNA and/or RNA or a protein) that is“objectively measured and evaluated as an indicator of normal biologicprocesses, pathogenic processes, or pharmacologic responses to atherapeutic intervention or other interventions”. Biomarkers alsoinclude any calculated indices created mathematically or combinations ofany one or more of the foregoing measurements, including temporal trendsand differences. Biomarkers may be measured at any level spatial ortemporal localization, including but not limited to within a tumor,within in a cell, or on the membrane of a cell.

By “agent” is meant any compound or composition suitable to beadministered in therapy, and explicitly includes chemical compounds;proteins, including antibodies or antigen-binding fragments thereof;peptides; and nucleic acid molecules.

By “subject” or “individual” is meant a human or non-human mammal,including, but not limited to, bovine, equine, canine, ovine, feline,and rodent, including murine and rattus, subjects. A “patient” is ahuman subject.

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder, and/or signs or symptomsassociated therewith, or slowing or halting the progression thereof. Itwill be appreciated that, although not precluded, treating a disorder orcondition does not require that the disorder, condition or symptomsassociated therewith be completely eliminated.

“Pre-treatment” means prior to the first administration of an IL-6antagonist according the methods described herein. Pre-treatment doesnot exclude, and often includes, the prior administration of treatmentsother than an IL-6 antagonist, such as treatment with a diuretic, suchas a loop diuretic.

By “biological sample” is meant any tissue, cell, fluid, or othermaterial derived from an organism (e.g., human subject). In certainembodiments, the biological sample is serum, plasma, urine, or wholeblood.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of a component of the invention in akit for detecting biomarkers disclosed herein. The instructionalmaterial of the kit of the invention can, for example, be affixed to acontainer which contains the component of the invention or be shippedtogether with a container which contains the component. Alternatively,the instructional material can be shipped separately from the containerwith the intention that the instructional material and the component beused cooperatively by the recipient.

The “level” of one or more biomarkers means the absolute or relativeamount or concentration of the biomarker in the sample as determined bymeasuring mRNA, cDNA, small organic molecules, nucleotides, ions orprotein, or any portion thereof such as oligonucleotide or peptide. Alevel of a biomarker may refer, based on context, to a global level or alevel within some subdivision of an organism or within a specificsample, by way of non-limiting example a level may refer to the amountor concentration of a biomarker in a urine sample or the level may referto the amount or concentration of the same biomarker in a plasma sample.

“Measuring” or “measurement,” or alternatively “detecting” or“detection,” means determining the presence, absence, quantity or amount(which can be an effective amount) of either a given substance within aclinical or subject-derived sample, including the derivation ofqualitative or quantitative concentration levels of such substances, orotherwise determining the values or categorization of a subject'sclinical parameters.

A “reference level” of a biomarker means a level of a biomarker that isindicative of the presence or absence of a particular phenotype orcharacteristic. When the level of a biomarker in a subject is above thereference level of the biomarker it is indicative of the presence of, orrelatively heightened level of, a particular phenotype orcharacteristic. When the level of a biomarker in a subject is below thereference level of the biomarker it is indicative of a lack of orrelative lack of a particular phenotype or characteristic.

8.1. Other Interpretational Conventions

Unless otherwise specified, antibody constant region residue numberingis according to the EU index as in Kabat.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. Ranges include the recited endpoints. Itshould be understood that the description in range format is merely forconvenience and brevity and should not be construed as an inflexiblelimitation on the scope of the invention. Accordingly, the descriptionof a range should be considered to have specifically disclosed all thepossible subranges as well as individual numerical values within thatrange. For example, description of a range such as from 1 to 6 should beconsidered to have specifically disclosed subranges such as from 1 to 3,from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., aswell as individual numbers within that range, for example, 1, 2, 2.7, 3,4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Unless specifically stated or apparent from context, as used herein theterm “or” is understood to be inclusive.

Unless specifically stated or apparent from context, as used herein, theterms “a”, “an”, and “the” are understood to be singular or plural. Thatis, the articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

In this disclosure, “comprises,” “comprising,” “containing,” “having,”“includes,” “including,” and linguistic variants thereof have themeaning ascribed to them in U.S. Patent law, permitting the presence ofadditional components beyond those explicitly recited.

Unless specifically stated or otherwise apparent from context, as usedherein the term “about” is understood as within a range of normaltolerance in the art, for example within 2 standard deviations of themean and is meant to encompass variations of ±20% or ±10%, morepreferably ±5%, even more preferably ±1%, and still more preferably±0.1% from the stated value.

Where an antibody equilibrium dissociation constant (K_(D)) is reported,K_(D) is determined by surface plasmon resonance with the antibody (orantigen-binding fragment thereof) fixed to the chip surface with ligandflowed thereover.

8.2. Summary of Experimental Observations

As further described below in Example 1, consecutive heart failure(“HF”) patients receiving high dose diuretic therapy at an outpatienttreatment center were enrolled in a prospective observational study.Plasma levels of IL-6 were measured to query systemic associations ofthis pro-inflammatory cytokine with various disease parameters, andurine levels of IL-6 were measured to query IL-6 associations with localinflammation and neurohormonal activation at the level of renal tissues.

Plasma and urine IL-6 levels were only modestly correlated with oneanother.

Increases in urine levels of IL-6 were significantly correlated in theseheart failure patients with measures of renal impairment, such asdiuretic resistance, lower estimated glomerular filtration rate(“eGFR”), and increased tissue-level renin-angiotensin-aldosteronesystem (“RAAS”) activation.

Although an inverse association between diuretic efficiency and plasmaIL-6 was also observed, upon adjustment for eGFR only urine IL-6remained significantly associated with risk of low diuretic efficiencyin these patients. Furthermore, when urine IL-6 and plasma IL-6 wereboth entered into a logistic regression model, only urine IL-6 remainedassociated with risk of low diuretic efficiency while plasma IL-6 showedno such association.

These data demonstrate that urine IL-6 level is a useful biomarker forrenal inflammation, and can be used to gauge renal dysfunction in thesetting of heart failure (cardiorenal syndrome). The data furthersuggest that serial measurements of urine IL-6 can be used to measurethe renal benefits of treatments administered to patients with heartfailure, notably heart failure patients with cardiorenal syndrome.

The urine IL-6 data, and to some extent the plasma IL-6 data, predictthat treatment with an IL-6 antagonist should be effective to reducerenal inflammation in heart failure patients, that is, to treat symptomsof cardiorenal syndrome. However, because infection is often aprecipitating cause of acute decompensation in heart failure patients,it is important to limit anti-cytokine and other immunosuppressivetherapies to those heart failure patients who are likely to respond withimproved renal and/or cardiac function. The cost of chronic IL-6antagonist therapy also militates for limiting treatment to those heartfailure patients who are likely to respond with improved renal and/orcardiac function.

As detailed below in Example 2, the analysis conducted in Example 1 wasexpanded to 129 patients. FIG. 1A is a bar graph showing diureticefficiency (“DE”) by tertiles of urine IL-6, confirming the inversecorrelation of urinary IL-6 level with diuretic efficiency observed inthe 98 patient subset. FIG. 1B is a bar graph showing diureticefficiency (“DE”) by tertiles of plasma IL-6 in these 129 patients,confirming an inverse correlation of plasma IL-6 levels with diureticefficiency.

The genotype of each of the 129 patients at the rs855791 singlenucleotide polymorphism (“SNP”) in transmembrane protease serine 6(“TMPRSS6”) was assessed. The TMPRSS6 polypeptide, also known asMatriptase-2 (MT2), cleaves hemojuvelin and inhibits bone morphogeneticprotein signaling. The rs855791 (G2321A) SNP alters the TMPRSS6 proteinsequence: the allele with highest frequency in the human population (themajor allele) is 2321G, encoding 736A; the allele with lowest frequencyin the human population (minor allele) is 2321A, encoding 736V.

As shown in FIG. 2A, urine levels of IL-6 were inversely correlated withdiuretic efficiency only in the patients having at least one copy of themajor allele of the TMPRSS6 rs855791 SNP (FIG. 2A, right panel,“AG+GG”); urine levels of IL-6 are not significantly correlated withdiuretic efficiency in patients homozygous for the minor allele (FIG.2A, left panel, “AA”).

As shown in FIG. 2B, plasma levels of IL-6 correlated inversely withdiuretic efficiency only in the patients having at least one copy of themajor allele of the TMPRSS6 rs855791 SNP (FIG. 2B, right panel,“AG+GG”); plasma levels of IL-6 are not significantly correlated withdiuretic efficiency in patients homozygous for the minor allele (FIG.2B, left panel, “AA”).

These data suggested that diuretic resistance (low diuretic efficiency)in heart failure patients could be treated with an IL-6 antagonist, butonly in those having at least one copy of the TMPRSS6 rs855791 majorallele.

In mouse M1 CCD cells, which are genotypically analogous to human cellshomozygous for the TMPRSS6 rs855791 major allele, the addition of IL-6correlated with the expression of ion transporters, NKCC2, ENaC-beta,and NCC. Increased expression of these ion transporters provides aputative mechanism for IL-6 mediated diuretic resistance. And becausethe increased expression is not known to be linked to hepcidinexpression, these data suggested that IL-6 antagonism could be effectivein treating diuretic resistance even in patients homozygous for theTMPRSS6 rs855791 minor allele.

Secondary analysis of data from two large clinical trials in differentheart failure patient populations confirmed the association of diureticresistance with IL-6 level (Example 5), independently of TMPRSS6rs855791 genotype (Example 6), providing evidence that IL-6 antagonismshould also be effective in treating diuretic resistance in patientshomozygous for the TMPRSS6 rs855791 minor allele.

8.3. Methods of Treating Diuretic Resistance

Accordingly, in a first aspect, methods are provided for treating apatient who requires diuresis but is resistant to diuretics. The methodscomprise administering, in combination with a diuretic, atherapeutically effective amount of an IL-6 antagonist to the patient.

In certain embodiments, the patient has elevated pre-treatment urinelevels of IL-6. In some embodiments, the patient has elevatedpre-treatment plasma IL-6 levels. In certain embodiments, the patienthas elevated pre-treatment levels of IL-6 in urine and in plasma.

In some embodiments, the patient has diuretic-resistant heart failure.In various embodiments, the patient has cardiorenal syndrome.

In some embodiments, the patient has been determined to have at leastone copy of the TMPRSS6 rs855791 major allele. In other embodiments, thepatient is homozygous for the TMPRSS6 rs855791 minor allele.

8.3.1. Diuretic Efficiency

In the methods described herein, the patient in need of the IL-6antagonist treatment has a disease or condition that requires diuresis,and is diuretic resistant.

In certain embodiments, the patient has been treated or is being treatedwith a thiazide diuretic, such as chlorothiazide (Diuril®),chlorthalidone, hydrochlorothiazide (Microzide®), indapamide, ormetolazone. In certain embodiments, the patient has been treated or isbeing treated with a loop diuretic, such as bumetanide (Bumex®),ethacrynic acid (Edecrin®), furosemide (Lasix®), or torsemide(Demadex®). In certain embodiments, the patient has been treated or isbeing treated with a potassium-sparing diuretic, such as amiloride,eplerenone (Inspra®), spironolactone (Aldactone®), or triamterene(Dyrenium®). In some embodiments, the patient has been treated or isbeing treated with more than one diuretic. In some embodiments, thepatient has been treated or is being treated with a plurality ofdifferent types of diuretics.

By definition, the patient resistant to diuretics has a low diureticefficiency. Diuretic efficiency is calculated as the increase in sodiumoutput per doubling of the loop diuretic dose, centered on a dose of 40mg of IV furosemide equivalents: diuretic efficiency=(mmol Naoutput)/(log₂(administered loop diuretic dose)−4.32). See Hanberg etal., Circ. Heart Fail. 2016; 9:e003180, the disclosure of which isincorporated herein by reference in its entirety.

In some embodiments, the patient has a diuretic efficiency of less than500, such as less than 450, 400, 350, 300, 250, or 200. In someembodiment, the patient has a diuretic efficiency of less than 200, suchas less than 195, 190, 185, 180, 175, 170, 165, 160, 155, or 150. Insome embodiments, the patient has a diuretic efficiency of less than150, such as less than 145, 140, 135, 130, 125, 120, 110, 105, or 100.In some embodiments, the patient has a diuretic efficiency of less than100, such as less than 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50. Inparticular embodiments, the patient has a diuretic efficiency of lessthan 50, such as less than 45, 40, 35, 30, or even less than 25, 20, 15,or 10.

In some embodiments, the patient resistant to diuretics requires adiuretic treatment of no less than 40 mg of furosemide (or equivalent)daily. In some of these embodiments, the patient requires a diuretictreatment of no less than 80 mg of furosemide (or equivalent) daily. Insome of these embodiments, the patient requires a diuretic treatment ofno less than 120 mg of furosemide (or equivalent) daily.

8.3.2. Pre-Treatment IL-6 Levels 8.3.2.1. Pre-Treatment Levels of IL-6in Urine

In certain embodiments, the patient has pre-treatment urine levels ofIL-6 of more than 5.0 pg IL-6/g creatinine, 6.0 pg IL-6/g creatinine,7.0 pg IL-6/g creatinine, 8.0 pg IL-6/g creatinine, 9.0 pg IL-6/gcreatinine, or 10.0 pg IL-6/g creatinine. In certain embodiments, thepatient has pre-treatment IL-6 urine levels of more than 11.0 pg IL-6/gcreatinine, 12.0 pg IL-6/g creatinine, 13.0 pg IL-6/g creatinine, 14.0pg IL-6/g creatinine, or 15.0 pg IL-6/g creatinine. In furtherembodiments, the patient has pre-treatment IL-6 levels in urine of morethan 16.0 pg IL-6/g creatinine, 17.0 pg IL-6/g creatinine, 18.0 pgIL-6/g creatinine, 19.0 pg IL-6/g creatinine, or 20.0 pg IL-6/gcreatinine. In particular embodiments, the patient has pre-treatmentIL-6 levels in urine of more than 21.0 pg IL-6/g creatinine, 22.0 pgIL-6/g creatinine, 23.0 pg IL-6/g creatinine, 24.0 pg IL-6/g creatinine,25.0 pg IL-6/g creatinine, 26.0 pg IL-6/g creatinine, 27.0 pg IL-6/gcreatinine, 28.0 pg IL-6/g creatinine, 29.0 pg IL-6/g creatinine, oreven more than 30.0 pg IL-6/g creatinine. In certain embodiments, thepatient has pre-treatment urine IL-6 levels of more than 35.0 pg/gcreatinine.

In certain embodiments, the patient has pre-treatment urine levels ofmore than 14.2 pg IL-6/g creatinine (“elevated urine IL-6 levels”). Inother embodiments, the patient has pre-treatment urine levels of lessthan 14.2 pg IL-/g creatinine.

In certain embodiments, the patient has levels of IL-6 in urine prior totreatment with an IL-6 antagonist and prior to treatment with a loopdiuretic of more than 5.0 pg IL-6/g creatinine, 6.0 pg IL-6/gcreatinine, 7.0 pg IL-6/g creatinine, 8.0 pg IL-6/g creatinine, 9.0 pgIL-6/g creatinine, or 10.0 pg IL-6/g creatinine. In some of theseembodiments, the patient has IL-6 urine levels of more than 11.0 pgIL-6/g creatinine, 12.0 pg IL-6/g creatinine, 13.0 pg IL-6/g creatinine,14.0 pg IL-6/g creatinine, or 15.0 pg IL-6/g creatinine. In furtherembodiments, the patient has levels of IL-6 in urine prior to treatmentwith an IL-antagonist and prior to treatment with a loop diuretic ofmore than 16.0 pg IL-6/g creatinine, 17.0 pg IL-6/g creatinine, 18.0 pgIL-6/g creatinine, 19.0 pg IL-6/g creatinine, or 20.0 pg IL-6/gcreatinine. In particular embodiments, the patient has IL-6 levels inurine of more than 21.0 pg IL-6/g creatinine, 22.0 pg IL-6/g creatinine,23.0 pg IL-6/g creatinine, 24.0 pg IL-6/g creatinine, 25.0 pg IL-6/gcreatinine, 26.0 pg IL-6/g creatinine, 27.0 pg IL-6/g creatinine, 28.0pg IL-6/g creatinine, 29.0 pg IL-6/g creatinine, or even more than 30.0pg IL-6/g creatinine. In certain embodiments, the patient has urine IL-6levels prior to treatment with an IL-antagonist and prior to treatmentwith a loop diuretic of more than 35.0 pg/g creatinine.

In certain embodiments, the patient has levels of IL-6 in urine prior totreatment with an IL-6 antagonist and prior to treatment with a loopdiuretic of more than 14.2 pg IL-6/g creatinine. In other embodiments,the patient has levels of IL-6 in urine prior to treatment with anIL-antagonist and prior to treatment with a loop diuretic of less than14.2 pg IL-/g creatinine.

8.3.2.2. Pre-Treatment Levels of IL-6 in Plasma

In various embodiments, the patient has elevated pre-treatment plasmaIL-6 levels.

In certain embodiments, the patient has pre-treatment plasma levels ofIL-6 of more than 2.0 pg/mL. In other embodiments, the patient haspre-treatment plasma levels of IL-6 of less than 2.0 pg/mL.

In certain embodiments, the patient has pre-treatment plasma levels ofIL-6 of more than 1.0 pg/ml, 1.1 pg/ml, 1.2 pg/ml, 1.3 pg/ml, 1.4 pg/ml,1.5 pg/ml, 1.6 pg/ml, 1.7 pg/ml, 1.8 pg/ml, 1.9 pg/ml, or 2.0 pg/ml. Incertain embodiments, the patient has pre-treatment plasma levels of IL-6of more than 2.1 pg/ml, 2.2 pg/ml, 2.3 pg/ml, 2.4 pg/ml, 2.5 pg/ml, 2.6pg/ml, 2.7 pg/ml, 2.8 pg/ml, 2.9 pg/ml, or 3.0 pg/ml. In certainembodiments, the patient has pre-treatment plasma levels of IL-6 of morethan 3.1 pg/ml, 3.2 pg/ml, 3.3 pg/ml, 3.4 pg/ml, 3.5 pg/ml, 3.6 pg/ml,3.7 pg/ml, 3.8 pg/ml, or 3.9 pg/ml.

In some embodiments, the patient has a pre-treatment IL-6 level ofgreater than 2 pg/mL, such as great than 3 pg/mL, 4 pg/mL, 5 pg/mL, 6pg/mL, 8 pg/mL, 10 pg/mL, 15 pg/mL, or 20 pg/mL. In certain embodiments,the patient has a pre-treatment IL-6 level of greater than 3 pg/mL. Incertain embodiments, the patient has a pre-treatment IL-6 level ofgreater than 5 pg/mL. In certain embodiments, the patient has apre-treatment IL-6 level of greater than 10 pg/mL.

8.3.2.3. Measurement of Pre-Treatment IL-6 Levels

Concentrations of IL-6 in urine, plasma, and serum can be determinedusing any standard assay known in the art. When IL-6 is measured inurine, the level may be indexed or normalized to another biomarker, incertain embodiments urinary creatinine.

In particular embodiments, concentrations are measured using theMesoScale Discovery (MSD) platform (Meso Scale diagnostics,Gaithersburg, Md., USA).

8.3.3. Heart Failure

In typical embodiments of the methods described herein, the patient hasheart failure.

In certain embodiments, the patient has NYHA functional class I heartfailure. In certain embodiments, the patient has NYHA functional classII heart failure. In certain embodiments, the patient has NYHAfunctional class III heart failure. In certain embodiments, the patienthas NYHA functional class IV heart failure.

In certain embodiments, the patient has acute heart failure. In certainembodiments, the patient has chronic heart failure.

In certain embodiments, the patient has a type of heart failure selectedfrom Table 1 below.

TABLE 1 ICD-10-CM Description I50 Heart failure Heart failurecomplicating abortion or ectopic or molar pregnancy, heart failurefollowing surgery, heart failure due to hypertension, heart failure dueto hypertension with chronic kidney disease, obstetic surgery andprocedures, rheumatic heart failure I50.9 Heart failure, unspecifiedBiventricular (heart) failure NOS Cardiac, heart or myocardial failureNOS Congestive heart disease Congestive heart failure Right ventricularfailure (secondary to left heart failure) I50.1 Left ventricular failureCardiac asthma Edema of lung with heart disease NOS Edema of lung withheart failure Left heart failure Pulmonary edema with heart disease NOSPulmonary edema with heart failure I50.20 Unspecified systolic(congestive) heart failure I50.21 Acute systolic (congestive) heartfailure I50.22 Chronic systolic (congestive) heart failure I50.23 Acuteon chronic systolic (congestive) heart failure I50.30 Unspecifieddiastolic (congestive) heart failure I50.31 Acute diastolic (congestive)heart failure I50.32 Chronic diastolic (congestive) heart failure I50.33Acute on chronic diastolic (congestive) heart failure I50.40 Unspecifiedcombined systolic (congestive) and diastolic (congestive) heart failureI50.41 Acute combined systolic (congestive) and diastolic (congestive)heart failure I50.42 Chronic combined systolic (congestive) anddiastolic (congestive) heart failure I50.43 Acute on chronic combinedsystolic (congestive) and diastolic (congestive) heart failure I50.1Left ventricular failure Heart failure, unspecified Biventricular(heart) failure NOS Cardiac, heart or myocardial failure NOS Congestiveheart disease Congestive heart failure Right ventricular failure(secondary to left heart failure)

In certain embodiments, the patient has cardiorenal syndrome. Inparticular embodiments, the patient has cardiorenal syndrome type 1. Inparticular embodiments, the patient has cardiorenal syndrome type 2. Inparticular embodiments, the patient has cardiorenal syndrome type 3. Inparticular embodiments, the patient has cardiorenal syndrome type 4.

In certain embodiments, the patient has diuretic resistant heartfailure. In certain of these embodiments, the heart failure patient hasa diuretic efficiency of less than 100, 95, 90, 85, 80, 75, 70, 65, 60,55, or 50. In particular embodiments, the patient has a diureticefficiency of less than 45, 40, 35, 30, or even less than 25, 20, 15, or10.

8.3.4. Kidney Disease

In some embodiments of methods described herein, the patient has kidneydisease.

In certain embodiments, the patient has diuretic resistant kidneydisease. In certain of these embodiments, the kidney disease patient hasa diuretic efficiency of less than 100, 95, 90, 85, 80, 75, 70, 65, 60,55, or 50. In particular embodiments, the patient has a diureticefficiency of less than 45, 40, 35, 30, or even less than 25, 20, 15, or10.

In particular embodiments, the patient has hepatorenal syndrome.

8.3.5. TMPRSS6 rs855791 Genotype

In certain embodiments, the patient has previously been determined tohave at least one copy of the TMPRSS6 rs855791 major allele. In otherembodiments, the method further comprises the earlier step ofdetermining that the patient has at least one copy of the TMPRSS6rs855791 major allele.

Preferably, the genotype at both alleles is determined, thus permittingidentification and discrimination of patients who are homozygous for theTMPRSS6 rs855791 major allele, heterozygous for the major and minorTMPRSS6 rs855791 alleles, and homozygous for the TMPRSS6 rs855791 minorallele.

The absence (major allele) or presence (minor allele) of SNP rs855791(2321G→A) in the TMPRSS6 gene is determined using standard techniques.

Typically, PCR is used to amplify a biological sample obtained from thepatient.

In certain embodiments, the absence or presence of polymorphism isdetected concurrently with amplification using real-time PCR (RT-PCR).In certain embodiments, the RT-PCR assay employs 5′ nuclease (TaqMan®probes), molecular beacons, and/or FRET hybridization probes. Reviewedin Espy et al., Clin. Microbiol. Rev. 2006 January; 19(1): 165-256,incorporated herein by reference in its entirety. In typicalembodiments, a commercially available assay is used. In selectembodiments, the commercially available assay is selected from the groupconsisting of TaqMan™ SNP Genotyping Assays (ThermoFisher); PCR SNPGenotyping Assay (Qiagen); Novallele Genotyping Assays (Canon); and SNPType™ assays (formerly SNPtype) (Fluidigm).

In certain embodiments, the absence or presence of polymorphism isdetected following amplification using hybridization with a probespecific for SNP rs855791, restriction endonuclease digestion, nucleicacid sequencing, primer extension, microarray or gene chip analysis,mass spectrometry, and/or a DNAse protection assay. In certainembodiments, the allelic variants are called by sequencing. In certainembodiments, Sanger sequencing is used. In certain embodiments, one of avariety of next-generation sequencing techniques is used, including forexample a sequencing technique selected from the group consisting ofmicroarray sequencing, Solexa sequencing (Illumina), Ion Torrent (LifeTechnologies), SOliD (Applied Biosystems), pyrosequencing,single-molecule real-time sequencing (Pacific Bio), nanopore sequencingand tunneling currents sequencing.

In certain embodiments, the absence or presence of polymorphisms isdetected using the procedures set forth in Example 2 below.

8.3.6. IL-6 Antagonists

The IL-6 antagonist used in the methods described herein is capable ofdecreasing the biological activity of IL-6.

8.3.6.1. Anti-IL-6 Antibodies

In certain embodiments, the IL-6 antagonist is an anti-IL-6 antibody orantigen-binding fragment or derivative thereof.

In certain embodiments, the IL-6 antagonist is a full-length anti-IL-6monoclonal antibody. In particular embodiments, the full-lengthmonoclonal antibody is an IgG antibody. In certain embodiments, thefull-length monoclonal antibody is an IgG1, IgG2, IgG3, or IgG4antibody. In certain embodiments, the IL-6 antagonist is a polyclonalcomposition comprising a plurality of species of full-length anti-IL-6antibodies, each of the plurality having unique CDRs. In certainembodiments, the IL-6 antagonist is an antibody fragment selected fromFab, Fab′, and F(ab′)2 fragments. In certain embodiments, the IL-6antagonist is a scFv, a disulfide-linked Fv (dsFv), or a single domainantibody, such as a camelid-derived VHH single domain Nanobody. Incertain embodiments, the IL-6 antagonist is immunoconjugate or fusioncomprising an IL-6 antigen-binding fragment. In certain embodiments, theantibody is bispecific or multispecific, with at least one of theantigen-binding portions having specificity for IL-6.

In certain embodiments, the antibody is fully human. In certainembodiments, the antibody is humanized. In certain embodiments, theantibody is chimeric and has non-human V regions and human C regiondomains. In certain embodiments, the antibody is murine.

In typical embodiments, the anti-IL-6 antibody has a K_(D) for bindinghuman IL-6 of less than 100 nM. In certain embodiments, the anti-IL-6antibody has a K_(D) for binding human IL-6 of less than 75 nM, 50 nM,25 nM, 20 nM, 15 nM, or 10 nM. In particular embodiments, the anti-IL-6antibody has a K_(D) for binding human IL-6 of less than 5 nM, 4 nM, 3nM, or 2 nM. In selected embodiments, the anti-IL-6 antibody has a K_(D)for binding human IL-6 of less than 1 nM, 750 pM, or 500 pM. In specificembodiments, the anti-IL-6 antibody has a K_(D) for binding human IL-6of no more than 500 pM, 400 pM, 300 pM, 200 pM, or 100 pM.

In typical embodiments, the anti-IL-6 antibody neutralizes thebiological activity of IL-6. In certain embodiments, the neutralizingantibody prevents binding of IL-6 to the IL-6 receptor.

In typical embodiments, the anti-IL-6 antibody has an eliminationhalf-life following intravenous administration of at least 7 days. Incertain embodiments, the anti-IL-6 antibody has an elimination half-lifeof at least 14 days, at least 21 days, or at least 30 days.

In certain embodiments, the anti-IL-6 antibody has a human IgG constantregion with at least one amino acid substitution that extends serumhalf-life as compared to the unsubstituted human IgG constant domain.

In certain embodiments, the IgG constant domain comprises substitutionsat residues 252, 254, and 256, wherein the amino acid substitution atamino acid residue 252 is a substitution with tyrosine, the amino acidsubstitution at amino acid residue 254 is a substitution with threonine,and the amino acid substitution at amino acid residue 256 is asubstitution with glutamic acid (“YTE”). See U.S. Pat. No. 7,083,784,incorporated herein by reference in its entirety. In certain extendedhalf-life embodiments, the IgG constant domain comprises substitutionsselected from T250Q/M428L (Hinton et al., J. Immunology 176:346-356(2006)); N434A (Yeung et al., J. Immunology 182:7663-7671 (2009)); orT307A/E380A/N434A (Petkova et al., International Immunology, 18:1759-1769 (2006)).

In certain embodiments, the elimination half-life of the anti-IL-6antibody is increased by utilizing the FcRN-binding properties of humanserum albumin. In certain embodiments, the antibody is conjugated toalbumin (Smith et al., Bioconjug. Chem., 12: 750-756 (2001)). In certainembodiments, the anti-IL-6 antibody is fused to bacterialalbumin-binding domains (Stork et al., Prot. Eng. Design Science 20:569-76 (2007)). In certain embodiments, the anti-IL-6 antibody is fusedto an albumin-binding peptide (Nguygen et al., Prot Eng Design Sel 19:291-297 (2006)). In certain embodiments, the anti-IL-antibody isbispecific, with one specificity being to IL-6, and one specificitybeing to human serum albumin (Ablynx, WO 2006/122825 (bispecificNanobody)).

In certain embodiments, the elimination half-life of the anti-IL-6antibody is increased by PEGylation (Melmed et al., Nature Reviews DrugDiscovery 7: 641-642 (2008)); by HPMA copolymer conjugation (Lu et al.,Nature Biotechnology 17: 1101-1104 (1999)); by dextran conjugation(Nuclear Medicine Communications, 16: 362-369 (1995)); by conjugationwith homo-amino-acid polymers (HAPs; HAPylation) (Schlapschy et al.,Prot Eng Design Sel 20: 273-284 (2007)); or by polysialylation(Constantinou et al., Bioconjug. Chem. 20: 924-931 (2009)).

8.3.6.1.1. MEDI5117 and Derivatives

In certain embodiments, the anti-IL-6 antibody or antigen-bindingportion thereof comprises all six CDRs of MEDI5117. In particularembodiments, the antibody or antigen-binding portion thereof comprisesthe MEDI5117 heavy chain V region and light chain V region. In specificembodiments, the antibody is the full-length MEDI5117 antibody. TheMEDI5117 antibody is described in WO 2010/088444 and US 2012/0034212,the disclosures of which are incorporated herein by reference in theirentireties. The MEDI5117 antibody has the following CDR and heavy andlight chain sequences:

MEDI5117 VH CDR1  (SEQ ID NO: 9) SNYMI  MEDI5117 VH CDR2 (SEQ ID NO: 10) DLYYYAGDTYYADSVKG  MEDI5117 VH CDR3  (SEQ ID NO: 11)WADDHPPWIDL  MEDI5117 VL CDR1  (SEQ ID NO: 12) RASQGISSWLA MEDI5117 VL CDR2  (SEQ ID NO: 13) KASTLES  MEDI5117 VL CDR3 (SEQ ID NO: 14) QQSWLGGS  MEDI5117 Heavy chain  (SEQ ID NO: 15)EVQLVESGGGLVQPGGSLRLSCAASGFTISSNYMIWVRQAPGKGLEWVSDLYYYAGDTYY ADSVKGRFTMSRDISKNTVYLQMNSLRAEDTAVYYCARWADDHPPWIDLWGRGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK  MEDI5117 Light chain  (SEQ ID NO: 16)DIQMTQSPSTLSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKVLIYKASTLESGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQSWLGGSFGQGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 

In certain embodiments, the anti-IL-6 antibody is a derivative ofMED5117.

In certain embodiments, the MED5117 derivative includes one or moreamino acid substitutions in the MED5117 heavy and/or light chain Vregions.

In certain embodiments, the derivative comprises fewer than 25 aminoacid substitutions, fewer than 20 amino acid substitutions, fewer than15 amino acid substitutions, fewer than 10 amino acid substitutions,fewer than 5 amino acid substitutions, fewer than 4 amino acidsubstitutions, fewer than 3 amino acid substitutions, fewer than 2 aminoacid substitutions, or 1 amino acid substitution relative to theoriginal V_(H) and/or V_(L) of the MEDI5117 anti-IL-6 antibody, whileretaining specificity for human IL-6.

In certain embodiments, the MED5117 derivative comprises an amino acidsequence that is at least 45%, at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% identical to the amino acidsequence of the V_(H) and V_(L) domain of MEDI5117. The percent sequenceidentity is determined using BLAST algorithms using default parameters.

In certain embodiments, the MED5117 derivative comprises an amino acidsequence in which the CDRs comprise an amino acid sequence that is atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 99% identical to the amino acid sequence of therespective CDRs of MEDI5117. The percent sequence identity is determinedusing BLAST algorithms using default parameters.

In certain embodiments, the V_(H) and/or V_(L) CDR derivatives compriseconservative amino acid substitutions at one or more predictednonessential amino acid residues (i.e., amino acid residues which arenot critical for the antibody to specifically bind to human IL-6).

8.3.6.1.2. Other Anti-IL-6 Antibodies

In certain embodiments, the anti-IL-6 antibody comprises the six CDRsfrom an antibody selected from the group consisting of siltuximab,gerilimzumab, sirukumab, clazakizumab, olokizumab, elsilimomab, VX30(VOP-R003; Vaccinex), EB-007 (EBI-029; Eleven Bio), ARGX-109 (ArGEN-X),FM101 (Femta Pharmaceuticals, Lonza) and ALD518/BMS-945429 (AlderBiopharmaceuticals, Bristol-Myers Squibb). In certain embodiments, theanti-IL-6 antibody comprises the heavy chain V region and light chain Vregion from an antibody selected from the group consisting ofsiltuximab, gerilimzumab, sirukumab, clazakizumab, olokizumab, VX30(VOP-R003; Vaccinex), EB-007 (EBI-029; Eleven Bio), ARGX-109 (ArGEN-X),FM101 (Femta Pharmaceuticals, Lonza) and ALD518/BMS-945429 (AlderBiopharmaceuticals, Bristol-Myers Squibb). In particular embodiments,the anti-IL-6 antibody is an antibody selected from the group consistingof siltuximab, gerilimzumab, sirukumab, clazakizumab, olokizumab, VX30(VOP-R003; Vaccinex), EB-007 (EBI-029; Eleven Bio), ARGX-109 (ArGEN-X),FM101 (Femta Pharmaceuticals, Lonza) and ALD518/BMS-945429 (AlderBiopharmaceuticals, Bristol-Myers Squibb).

In certain embodiments, the anti-IL-6 antibody comprises the six CDRsfrom an antibody selected from those described in US 2016/0168243, US2016/0130340, US 2015/0337036, US 2015/0203574, US 2015/0140011, US2015/0125468, US 2014/0302058, US 2014/0141013, US 2013/0280266, US2013/0017575, US 2010/0215654, US 2008/0075726, U.S. Pat. No. 5,856,135,US 2006/0240012, US 2006/0257407, or U.S. Pat. No. 7,291,721, thedisclosures of which are incorporated herein by reference in theirentireties.

8.3.6.2. Anti-IL-6 Receptor Antibodies

In certain embodiments, the IL-6 antagonist is an anti-IL-6 receptorantibody or antigen-binding fragment or derivative thereof.

In certain embodiments, the IL-6 antagonist is a full-length anti-IL-6receptor monoclonal antibody. In particular embodiments, the full-lengthmonoclonal antibody is an IgG antibody. In certain embodiments, thefull-length monoclonal antibody is an IgG1, IgG2, IgG3, or IgG4antibody. In certain embodiments, the IL-6 antagonist is a polyclonalcomposition comprising a plurality of species of full-length anti-IL-6receptor antibodies, each of the plurality having unique CDRs. Incertain embodiments, the IL-6 antagonist is an antibody fragmentselected from Fab and Fab′ fragments. In certain embodiments, the IL-6antagonist is a scFv, a single domain antibody, including acamelid-derived VHH single domain Nanobody. In certain embodiments, theantibody is bispecific or multispecific, with at least one of theantigen-binding portions having specificity for IL-6R.

In certain embodiments, the antibody is fully human. In certainembodiments, the antibody is humanized. In certain embodiments, theantibody is chimeric and has non-human V regions and human C regiondomains. In certain embodiments, the antibody is murine.

In typical embodiments, the anti-IL-6 receptor antibody has a K_(D) forbinding human IL-6R of less than 100 nM. In certain embodiments, theanti-IL-6R antibody has a K_(D) for binding human IL-6R of less than 75nM, 50 nM, 25 nM, 20 nM, 15 nM, or 10 nM. In particular embodiments, theanti-IL-6 receptor antibody has a K_(D) for binding human IL-6R of lessthan 5 nM, 4 nM, 3 nM, or 2 nM. In selected embodiments, the anti-IL-6receptor antibody has a K_(D) for binding human IL-6R of less than 1 nM,750 pM, or 500 pM. In specific embodiments, the anti-IL-6 receptorantibody has a K_(D) for binding human IL-6R of no more than 500 pM, 400pM, 300 pM, 200 pM, or 100 pM.

In typical embodiments, the anti-IL-6R reduces the biological activityof IL-6.

In typical embodiments, the anti-IL-6R antibody has an eliminationhalf-life following intravenous administration of at least 7 days. Incertain embodiments, the anti-IL-6R antibody has an eliminationhalf-life of at least 14 days, at least 21 days, or at least 30 days.

In certain embodiments, the anti-IL-6R antibody has a human IgG constantregion with at least one amino acid substitution that extends serumhalf-life as compared to the unsubstituted human IgG constant domain.

In certain embodiments, the IgG constant domain comprises substitutionsat residues 252, 254, and 256, wherein the amino acid substitution atamino acid residue 252 is a substitution with tyrosine, the amino acidsubstitution at amino acid residue 254 is a substitution with threonine,and the amino acid substitution at amino acid residue 256 is asubstitution with glutamic acid (“YTE”). See U.S. Pat. No. 7,083,784,incorporated herein by reference in its entirety. In certain extendedhalf-life embodiments, the IgG constant domain comprises substitutionsselected from T250Q/M428L (Hinton et al., J. Immunology 176:346-356(2006)); N434A (Yeung et al., J. Immunology 182:7663-7671 (2009)); orT307A/E380A/N434A (Petkova et al., International Immunology, 18:1759-1769 (2006)).

In certain embodiments, the elimination half-life of the anti-IL-6Rantibody is increased by utilizing the FcRN-binding properties of humanserum albumin. In certain embodiments, the antibody is conjugated toalbumin (Smith et al., Bioconjug. Chem., 12: 750-756 (2001)). In certainembodiments, the anti-IL-6R antibody is fused to bacterialalbumin-binding domains (Stork et al., Prot. Eng. Design Science 20:569-76 (2007)). In certain embodiments, the anti-IL-6 antibody is fusedto an albumin-binding peptide (Nguygen et al., Prot Eng Design Sel 19:291-297 (2006)). In certain embodiments, the anti-IL-antibody isbispecific, with one specificity being to IL-6R, and one specificitybeing to human serum albumin (Ablynx, WO 2006/122825 (bispecificNanobody)).

In certain embodiments, the elimination half-life of the anti-IL-6Rantibody is increased by PEGylation (Melmed et al., Nature Reviews DrugDiscovery 7: 641-642 (2008)); by HPMA copolymer conjugation (Lu et al.,Nature Biotechnology 17: 1101-1104 (1999)); by dextran conjugation(Nuclear Medicine Communications, 16: 362-369 (1995)); by conjugationwith homo-amino-acid polymers (HAPs; HAPylation) (Schlapschy et al.,Prot Eng Design Sel 20: 273-284 (2007)); or by polysialylation(Constantinou et al., Bioconjug. Chem. 20: 924-931 (2009)).

In certain embodiments, the anti-IL-6R antibody or antigen-bindingportion thereof comprises all six CDRs of tocilizumab. In particularembodiments, the antibody or antigen-binding portion thereof comprisesthe tocilizumab heavy chain V region and light chain V region. Inspecific embodiments, the antibody is the full-length tocilizumabantibody.

In certain embodiments, the anti-IL-6R antibody or antigen-bindingportion thereof comprises all six CDRs of sarilumab. In particularembodiments, the antibody or antigen-binding portion thereof comprisesthe sarilumab heavy chain V region and light chain V region. In specificembodiments, the antibody is the full-length sarilumab antibody.

In certain embodiments, the anti-IL-6R antibody or antigen-bindingportion thereof comprises all six CDRs of VX30 (Vaccinex), ARGX-109(arGEN-X), FM101 (Formatech), SA237 (Roche), NI-1201 (NovImmune), or anantibody described in US 2012/0225060.

In certain embodiments, the anti-IL-6R antibody or antigen-bindingportion thereof is a single domain antibody. In particular embodiments,the single domain antibody is a camelid VHH single domain antibody. Inspecific embodiments, the antibody is vobarilizumab (ALX-0061) (AblynxNV).

8.3.6.3. Anti-IL-6:IL-6R Complex Antibodies

In certain embodiments, the IL-6 antagonist is an antibody specific forthe complex of IL-6 and IL-6R. In certain embodiments, the antibody hasthe six CDRs of an antibody selected from those described in US2011/0002936, which is incorporated herein by reference in its entirety.

8.3.6.4. JAK and STAT Inhibitors

IL-6 is known to signal via the JAK-STAT pathway.

In certain embodiments, the IL-6 antagonist is an inhibitor of the JAKsignaling pathway. In certain embodiments, the JAK inhibitor is aJAK1-specific inhibitor. In certain embodiments, the JAK inhibitor is aJAK3-specific inhibitor. In certain embodiments, the JAK inhibitor is apan-JAK inhibitor.

In certain embodiments, the JAK inhibitor is selected from the groupconsisting of tofacitinib (Xeljanz), decernotinib, ruxolitinib,upadacitinib, baricitinib, filgotinib, lestaurtinib, pacritinib,peficitinib, INCB-039110, ABT-494, INCB-047986 and AC-410.

In certain embodiments, the IL-6 antagonist is a STAT3 inhibitor. In aspecific embodiment, the inhibitor is AZD9150 (AstraZeneca, IsisPharmaceuticals), a STAT3 antisense molecule.

8.3.6.5. Additional IL-6 Antagonists

In certain embodiments, the IL-6 antagonist is an antagonist peptide.

In certain embodiments, the IL-6 antagonist is C326 (an IL-6 antagonistby Avidia, also known as AMG220), or FE301, a recombinant proteininhibitor of IL-6 (Ferring International Center S.A., Conaris ResearchInstitute AG). In certain embodiments, the anti-IL-6 antagonistcomprises soluble gp130, FE301 (Conaris/Ferring).

8.3.7. Dosage Regimens 8.3.7.1. Antibodies, Antigen-Binding Fragments,Peptides

In typical embodiments, antibody, antigen-binding fragments, and peptideIL-6 antagonists are administered parenterally.

In some parenteral embodiments, the IL-6 antagonist is administeredintravenously. In certain intravenous embodiments, the IL-6 antagonistis administered as a bolus. In certain intravenous embodiments, the IL-6antagonist is administered as an infusion. In certain intravenousembodiments, the IL-6 antagonist is administered as a bolus followed byinfusion. In some parenteral embodiments, the IL-6 antagonist isadministered subcutaneously.

In certain embodiments, the antibody, antigen-binding fragment, orpeptide IL-6 antagonist is administered in a dose that is independent ofpatient weight or surface area (flat dose).

In certain embodiments, the intravenous flat dose is 1 mg, 2 mg, 3 mg, 4mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg. In certain embodiments, theintravenous flat dose is 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17mg, 18 mg, 19 mg, or 20 mg. In certain embodiments, the intravenous flatdose is 25 mg, 30 mg, 40 mg, or 50 mg. In certain embodiments, theintravenous flat dose is 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg. Incertain embodiments, the intravenous flat dose is 1-10 mg, 10-15 mg,15-20 mg, 20-30 mg, 30-40 mg, or 40-50 mg. In certain embodiments, theintravenous flat dose is 1-40 mg, or 50-100 mg.

In certain embodiments, the subcutaneous flat dose is 10 mg, 20 mg, 30mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg. In certainembodiments, the subcutaneous flat dose is 110 mg, 120 mg, 130 mg, 140mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, or 200 mg. In certainembodiments, the subcutaneous flat dose is 210 mg, 220 mg, 230 mg, 240mg, or 250 mg. In certain embodiments, the subcutaneous flat dose is10-100 mg, 100-200 mg, or 200-250 mg. In certain embodiments, thesubcutaneous flat dose is 10-20 mg, 20-30 mg, 30-40 mg, 40-50 mg, 50-60mg, 60-70 mg, 70-80 mg, 80-90 mg, or 90-100 mg. In certain embodiments,the subcutaneous flat dose is 100-125 mg, 125-150 mg, 150-175 mg,175-200 mg, or 200-250 mg.

In certain embodiments, the antibody, antigen-binding fragment, orpeptide IL-6 antagonist is administered as a patient weight-based dose.

In certain embodiments, the antagonist is administered at an intravenousdose of 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg or 1.0 mg/kg. In certainembodiments, the antagonist is administered at a dose of 1.5 mg/kg, 2mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, or 5 mg/kg.

In certain embodiments, the subcutaneous weight-based dose is 0.1 mg/kg,0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8mg/kg, 0.9 mg/kg or 1.0 mg/kg. In certain embodiments, the antagonist isadministered at a dose of 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5mg/kg, 4 mg/kg, 4.5 mg/kg, or 5 mg/kg.

In various intravenous embodiments, the IL-6 antagonist is administeredonce every 7 days, once every 14 days, once every 21 days, once every 28days, or once a month. In various subcutaneous embodiments, the IL-6antagonist is administered once every 14 days, once every 28 days, oncea month, once every two months (every other month), or once every threemonths.

In certain preferred embodiments, the IL-6 antagonist is the MEDI5117antibody. In certain embodiments, MEDI5117 is administered in a flatdose of 1-30 mg IV once every week. In certain embodiments, the MEDI5117antibody is administered in a flat dose of 1, 2, 3, 4, 5, 7.5, 10, 15,20, 25, or 30 mg IV once every week. In certain embodiments, theMEDI5117 antibody is administered in a flat dose of 25-250 mg s.c. onceevery month to once every three months. In particular embodiments,MEDI5117 is administered at a dose of 30 mg, 45 mg, 60 mg, 75 mg, 100mg, 120 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 240 mg, or 250 mgs.c. once every month, once every two months, or once every 3 months.

In certain embodiments, the IL-6 antagonist is tocilizumab. In certainembodiments, tocilizumab is administered s.c. in a starting dose forpatients ≥100 kg of 162 mg once every week. In certain embodiments,tocilizumab is administered intravenously at a dose of 4 mg/kg onceevery 4 weeks followed by an increase to 8 mg/kg every 4 weeks based onclinical response.

8.3.7.2. JAK and STAT Inhibitors

In typical embodiments, small molecule JAK inhibitors and STATinhibitors are administered orally.

In certain embodiments, the inhibitor is administered once or twice aday at an oral dose of 1-10 mg, 10-20 mg, 20-30 mg, 30-40 mg, or 40-50mg. In certain embodiments, the inhibitor is administered once or twicea day at a dose of 50-60 mg, 60-70 mg, 70-80 mg, 80-90 mg, or 90-100 mg.In certain embodiments, the inhibitor is administered at a dose of 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 mg PO once or twice a day. Incertain embodiments, the inhibitor is administered at a dose of 75 mg POQD or BID, 100 mg PO QD or BID.

In certain embodiments, the JAK inhibitor is tofacitinib, and isadministered at a dose of 5 mg PO BID or 11 mg PO qDay,

In certain embodiments, the JAK inhibitor is decernotinib, and isadministered at a dose of 25 mg, 50 mg, 100 mg, or 150 mg PO BID.

In certain embodiments, the inhibitor is ruxolitinib, and isadministered at dose of 25 mg PO BID, 20 mg PO BID, 15 mg PO BID, 10 mgPO BID, or 5 mg PO BID.

8.3.7.3. Treating to Goal

In certain embodiments, the IL-6 antagonist is administered at a dose,on a schedule, and for a period sufficient to increase diureticefficiency. In certain of these embodiments, the IL-6 antagonist isadministered at a dose, on a schedule, and for a period sufficient toincrease diuretic efficiency to normal levels.

In certain embodiments, the IL-6 antagonist is administered at a dose,on a schedule, and for a period sufficient to increase eGFR, and incertain of these embodiments, the IL-6 antagonist is administered at adose, on a schedule, and for a period sufficient to increase eGFR tonormal levels.

8.3.7.4. IL-6 Level Monitoring

In certain embodiments, the impact of treatment with IL-6 antagonists oncardiorenal parameters may be monitored by measuring a level of IL-6 ina urine or plasma sample from the patient. It is specificallycontemplated that the methods of the invention may be used to monitorthe efficacy of treatments for cardiorenal syndrome and may motivate,for example, a change of dose or therapeutic.

Accordingly, in certain embodiments, the method further comprises thesubsequent step of determining the level of IL-6 in urine, in plasma, orin both urine and plasma. In certain of these embodiments, the methodfurther comprises a final step of adjusting the dose of at least onesubsequent administration of IL-6 antagonist based on IL-6 leveldetermined in the immediately preceding step.

8.3.8. Additional Therapeutic Agents

In certain embodiments of the methods described herein, the methodfurther comprises administering at least one therapeutic agentadditional to the IL-6 antagonist, wherein the additional therapeuticagent treats one or more cardiovascular or renal symptoms of heartfailure. Specific treatment will be determined on a case by case basisby the attending physician.

8.3.8.1. Standard Heart Failure Agents

In certain embodiments, the additional therapeutic agent is a diuretic.

In particular embodiments, the diuretic is a loop diuretic. In selectembodiments, the loop diuretic is selected from the group consisting offurosemide, torsemide, bumetanide, and ethacrynic acid. In particularembodiments, the loop diuretic is furosemide. In certain embodiments,furosemide is administered orally. In certain embodiments, furosemide isadministered intravenously. In certain embodiments, the diuretic is athiazide diuretic. In particular embodiments, the thiazide diuretic ischlorothiazide, hydrochlorothiazide, chlorthalidone, indapamide, ormetolazone. In certain embodiments, the diuretic is a potassium sparingdiuretic.

In certain embodiments, the additional therapeutic agent is an ACEinhibitor. In certain embodiments, the ACE inhibitor is selected fromthe group consisting of benazepril, captopril, enalapril, fosinopril,lisinopril, moexipril, perindopril, quinapril, ramipril andtrandolapril.

In certain embodiments, the additional therapeutic agent is anangiotensin receptor blocker (“ARB”). In certain embodiments, the ARB iseprosartan, olmesartan, valsartan, telmisartan, losartan, azilsartanmedoxomil, candesartan, or irbesartan.

In certain embodiments, the additional therapeutic agent is a β-blocker,a calcium antagonist, or a mineralocorticoid receptor antagonist.

In certain embodiments, the additional therapeutic agent is anatriuretic peptide, such as a B-type natriuretic peptide or N-terminalpro-B-type natriuretic peptide.

In certain embodiments, the additional therapeutic agent is an adenosineantagonist, such as rolofylline.

Specific treatment will be determined on a case-by-case basis by theattending physician.

8.3.8.2. Nitroxyl Donors

In certain embodiments, the additional therapeutic agent is a nitroxyldonor, and the method further comprises administering a therapeuticallyeffective amount of the nitroxyl donor.

In particular embodiments, the nitroxyl donor is selected from thecompounds described in one or more of U.S. Pat. Nos. 9,499,511;9,487,498; 9,464,061; 9,458,127; 9,221,780; 9,181,213; 9,156,804;9,115,064; 9,018,411; 8,987,326; RE45,314; 8,674,132; 8,227,639; and8,030,356, the disclosures of which are incorporated herein by referencein their entireties.

In selected embodiments, the nitroxyl donor is selected from thecompounds described in U.S. Pat. No. RE45,314. In specific embodiments,the nitroxyl donor is selected from the compounds described in U.S. Pat.No. 9,156,804.

8.3.8.3. Sodium-Free Chloride Supplementation

In certain embodiments, the additional therapeutic agent is a sodiumfree chloride salt. In certain embodiments, the agent is lysinechloride.

8.4. Methods of Improving Treatment of Heart Failure

In another aspect, methods are provided for improving treatment of heartfailure by discontinuing therapy that is ineffective, thereby reducingside effects and reducing cost without loss of treatment efficacy. Themethods comprise discontinuing administration of an IL-6 antagonist to apatient with heart failure, wherein the patient has been determined tobe homozygous for the TMPRSS6 rs855791 minor allele. In one series ofembodiments, the patient has previously been determined to be homozygousfor the TMPRSS6 rs855791 minor allele. In another series of embodiments,the method further comprises the earlier step of determining that thepatient is homozygous for the TMPRSS6 rs855791 minor allele.

In certain embodiments, the patient has elevated pre-treatment urinelevels of IL-6. In certain embodiments, the patient has elevatedpre-treatment plasma levels of IL-6. In certain embodiments, the patienthas elevated pre-treatment urine and elevated pre-treatment plasmalevels of IL-6.

In particular embodiments, the patient has cardiorenal syndrome.

8.5. Diagnostic, Prognostic, and Treatment Guidance Methods

In another aspect, methods are provided for determining if a subjectwould benefit from IL-6 antagonist treatment for heart failure. Themethod comprises measuring a level of IL-6 in a urine sample or plasmasample from the subject, comparing the measured level of IL-6 to apredetermined reference level, and determining whether or not themeasured level of IL-6 is greater than the corresponding referencelevel, wherein when the measured level of IL-6 is greater than thecorresponding reference level, IL-6 antagonist treatment is recommended.

IL-6 may be measured in plasma or in urine according to the methods setforth in Section 6.3.2.3 above. Reference levels for IL-6 in urine andplasma may be determined by measuring IL-6 levels in a referencepopulation. A person of skill in the art is able to determine areference level for the level of a biomarker in a population based onclinical experience and common levels of the biomarker in samples fromthe population.

In further aspects, methods are provided for determining if a subject isin need of IL-6 antagonist treatment for impaired glomerular filtration,low diuretic efficiency, high urine angiotensin, high plasma renin, oris at risk of mortality due to cardiorenal syndrome using the abovedescribed method. As shown in FIG. 3 and discussed in Example 1hereinbelow, high levels of IL-6 in urine or plasma are associated withthese parameters.

8.6. Kits

In a further aspect, a kit is provided. In general, kits will comprisedetection reagents that are suitable for detecting the presence ofbiomarkers of interest and with instructions for use in accordance withthe methods of the invention. The kit may comprise antibodies or otherimmunohistochemical reagents capable of binding to IL-6. The kit maycontain capture and detection antibodies suitable for performing anELISA for measuring IL-6 in urine or plasma. In certain embodiments thekit may contain tools and reagents for preparing urine and plasmasamples for the ELISA or for indexing IL-6 in urine to the concentrationof another biomarker, in certain embodiments IL-6 is indexed againstcreatinine.

8.7. Further Embodiments

Further embodiments are provided in the following numbered embodiments.

1. A method of treating heart failure, comprising:

administering a therapeutically effective amount of an IL-6 antagonistto a patient with heart failure,

wherein the patient has been determined to have at least one copy of theTMPRSS6 rs855791 major allele.

2. The method of embodiment 1, wherein the patient has previously beendetermined to have at least one copy of the TMPRSS6 rs855791 majorallele.

3. The method of embodiment 1, further comprising the earlier step of:

determining that the patient has at least one copy of the TMPRSS6rs855791 major allele.

4. The method of any one of embodiments 1-3, wherein the patient haselevated pre-treatment urine levels of IL-6.

5. The method of any one of embodiments 1-4, wherein the patient haselevated pre-treatment plasma levels of IL-6.

6. The method of any one of embodiments 1-5, wherein the patient hasacute heart failure.

7. The method of any one of embodiments 1-5, wherein the patient haschronic heart failure.

8. The method of any one of embodiments 1-7, wherein the patient hascardiorenal syndrome.

9. The method of embodiment 8, wherein the patient has cardiorenalsyndrome type 4.

10. The method of any one of embodiments 1-9, wherein the patient hasdiuretic resistant heart failure.

11. The method of embodiment 10, wherein the patient's diureticefficiency is less than 95.

12. The method of embodiment 11, wherein the patient's diureticefficiency is less than 90.

13. The method of embodiment 12, wherein the patient's diureticefficiency is less than 85.

14. The method of embodiment 13, wherein the patient's diureticefficiency is less than 80.

15. The method of embodiment 14, wherein the patient's diureticefficiency is less than 75.

16. The method of embodiment 15, wherein the patient's diureticefficiency is less than 70.

17. The method of embodiment 16, wherein the patient's diureticefficiency is less than 65.

18. The method of any one of embodiments 1-17, wherein the IL-6antagonist is an anti-IL-6 antibody, or antigen-binding fragment orderivative thereof.

19. The method of embodiment 18, wherein the anti-IL-6 antibody orantigen-binding fragment or derivative has a K_(D) for binding humanIL-6 of less than 100 nM.

20. The method of embodiment 19, wherein the antibody or antigen-bindingfragment or derivative has a K_(D) for binding human IL-6 of less than50 nM.

21. The method of embodiment 20, wherein the antibody or antigen-bindingfragment or derivative has a K_(D) for binding human IL-6 of less than10 nM.

22. The method of embodiment 21, wherein the antibody or antigen-bindingfragment or derivative has a K_(D) for binding human IL-6 of less than 1nM.

23. The method of any one of embodiments 18-22, wherein the anti-IL-6antibody or antigen-binding fragment or derivative has an eliminationhalf-life following intravenous administration of at least 7 days.

24. The method of embodiment 23, wherein the anti-IL-6 antibody orantigen-binding fragment or derivative has an elimination half-lifefollowing intravenous administration of at least 14 days.

25. The method of embodiment 24, wherein the anti-IL-6 antibody orantigen-binding fragment or derivative has an elimination half-lifefollowing intravenous administration of at least 21 days.

26. The method of embodiment 25, wherein the anti-IL-6 antibody orantigen-binding fragment or derivative has an elimination half-lifefollowing intravenous administration of at least 30 days.

27. The method of any one of embodiments 18-26, wherein the IL-6antagonist is a full-length monoclonal anti-IL-6 antibody.

28. The method of embodiment 27, wherein the antibody is an IgG1 or IgG4antibody.

29. The method of embodiment 28, wherein the antibody is an IgG1antibody.

30. The method of any one of embodiments 18-29, wherein the anti-IL-6antibody or antigen-binding fragment or derivative is fully human.

31. The method of any one of embodiments 18-29, wherein the anti-IL-6antibody or antigen-binding fragment or derivative is humanized.

32. The method of any one of embodiments 18-31, wherein the anti-IL-6antibody or antigen-binding fragment or derivative comprises all sixvariable region CDRs of MED5117.

33. The method of embodiment 32, wherein the antibody comprises the VHand VL of MED5117.

34. The method of embodiment 33, wherein the antibody is MED5117.

35. The method of any one of embodiments 18-31, wherein the anti-IL-6antibody or antigen-binding fragment or derivative comprises all sixvariable region CDRs of an antibody selected from the group consistingof siltuximab, gerilimzumab, sirukumab, clazakizumab, olokizumab,elsilimomab, VX30 (VOP-R003; Vaccinex), EB-007 (EBI-029; Eleven Bio),ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza) andALD518/BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb).36. The method of embodiment 35, wherein the anti-IL-6 antibody orantigen-binding fragment or derivative comprises the heavy chain Vregion and light chain V region from an antibody selected from the groupconsisting of siltuximab, gerilimzumab, sirukumab, clazakizumab,olokizumab, VX30 (VOP-R003; Vaccinex), EB-007 (EBI-029; Eleven Bio),ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza) andALD518/BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb).37. The method of embodiment 36, wherein the anti-IL-6 antibody orantigen-binding fragment or derivative is an antibody selected from thegroup consisting of siltuximab, gerilimzumab, sirukumab, clazakizumab,olokizumab, VX30 (VOP-R003; Vaccinex), EB-007 (EBI-029; Eleven Bio),ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza) andALD518/BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb).38. The method of any one of embodiments 18-26, wherein the IL-6antagonist is a single domain antibody, a Vim Nanobody, an Fab, or ascFv.39. The method of any one of embodiments 1-17, wherein the IL-6antagonist is an anti-IL-6R antibody, or antigen-binding fragment orderivative thereof.40. The method of embodiment 39, wherein the anti-IL-6R antibody,antigen-binding fragment, or derivative comprises the 6 CDRs oftocilizumab.41. The method of embodiment 39, wherein the anti-IL-6R antibody,antigen-binding fragment, or derivative comprises the 6 CDRs ofvobarilizumab.42. The method of any one of embodiments 1-17, wherein the IL-6antagonist is a JAK inhibitor.43. The method of embodiment 42, wherein the JAK inhibitor is selectedfrom the group consisting of tofacitinib (Xeljanz), decernotinib,ruxolitinib, upadacitinib, baricitinib, filgotinib, lestaurtinib,pacritinib, peficitinib, INCB-039110, ABT-494, INCB-047986 and AC-410.44. The method of any one of embodiments 1-17, wherein the IL-6antagonist is a STAT3 inhibitor.45. The method of any one of embodiments 18-41, wherein the IL-6antagonist is administered parenterally.46. The method of embodiment 45, wherein the IL-6 antagonist isadministered subcutaneously.47. The method of any one of embodiments 42 or 43, wherein the IL-6antagonist is administered orally.48. The method of any one of embodiments 1-47, wherein the IL-6antagonist is administered at a dose, on a schedule, and for a periodsufficient to increase diuretic efficiency.49. The method of embodiment 48, wherein the IL-6 antagonist isadministered at a dose, on a schedule, and for a period sufficient toincrease diuretic efficiency to normal levels.50. The method of any one of embodiments 1-49, wherein the IL-6antagonist is administered at a dose, on a schedule, and for a periodsufficient to increase eGFR.51. The method of embodiment 50, wherein the IL-6 antagonist isadministered at a dose, on a schedule, and for a period sufficient toincrease eGFR to normal levels.52. The method of any one of embodiments 1-51, further comprising thesubsequent step of determining the level of IL-6 in urine.53. The method of any one of embodiments 1-51, further comprising thesubsequent step of determining the level of IL-6 in plasma.54. The method of any one of embodiments 1-51, further comprising thesubsequent step of determining the level of IL-6 in urine and in plasma.55. The method of any one of embodiments 52-54, further comprising afinal step of adjusting the dose of IL-6 antagonist for subsequentadministration based on IL-6 level determined in the immediatelypreceding step.56. A method of determining if a subject requires treatment forcardiorenal syndrome, the method comprising:

-   -   a) measuring a level of IL-6 in a urine or plasma sample from        the subject,    -   b) comparing the measured level of IL-6 to a predetermined        reference level,    -   c) determining whether or not the measured level of IL-6 is        greater than the corresponding reference level, wherein when the        measured level of IL-6 is greater than the corresponding        reference level, the subject has cardiorenal syndrome, and        treatment is recommended.        57. A method of determining if a subject requires treatment for        impaired glomerular filtration, comprising:    -   a) measuring a level of IL-6 in a urine sample from the subject,    -   b) comparing the measured level of IL-6 to a predetermined        reference level,    -   c) determining whether or not the measured level of IL-6 is        greater than the corresponding reference level,    -   wherein when the measured level of IL-6 is greater than the        corresponding reference level, the subject has impaired        glomerular filtration, and treatment is recommended.        58. A method of determining if a subject requires treatment for        low diuretic efficiency, comprising:    -   a) measuring a level of IL-6 in a urine sample from the subject,    -   b) comparing the measured level of IL-6 to a predetermined        reference level,    -   c) determining whether or not the measured level of IL-6 is        greater than the corresponding reference level,    -   wherein when the measured level of IL-6 is greater than the        corresponding reference level, the subject has low diuretic        efficiency, and treatment is recommended.        59. A method of determining if a subject requires treatment for        high urine angiotensin, comprising:    -   a) measuring a level of IL-6 in a urine sample from the subject,    -   b) comparing the measured level of IL-6 to a predetermined        reference level,    -   c) determining whether or not the measured level of IL-6 is        greater than the corresponding reference level,    -   wherein when the measured level of IL-6 is greater than the        corresponding reference level, the subject has high urine        angiotensin, and treatment is recommended.        60. A method of determining if a subject requires treatment for        high plasma renin, comprising:    -   a) measuring a level of IL-6 in a plasma sample from the        patient,    -   b) comparing the measured level of IL-6 to a predetermined        reference level,    -   c) determining whether or not the measured level of IL-6 is        greater than the corresponding reference level,    -   wherein when the measured level of IL-6 is greater than the        corresponding reference level, the subject has high plasma        renin, and treatment is recommended.        61. A method of determining if a subject is at risk of mortality        due to cardiorenal syndrome and requires treatment, comprising:    -   a) measuring a level of IL-6 in a plasma sample from the        patient,    -   b) comparing the measured level of IL-6 to a predetermined        reference level,    -   c) determining whether or not the measured level of IL-6 is        greater than the corresponding reference level,    -   wherein when the measured level of IL-6 is greater than the        corresponding reference level, the subject is at risk of        mortality due to cardiorenal syndrome, and treatment is        recommended.        62. The method of any one of embodiments 56-61, wherein the        patient is a heart failure patient.        63. The method of any of embodiments 56-62, wherein the level of        IL-6 is measured using an enzyme-linked immunosorbent assay        (ELISA).        64. A method of treating cardiorenal syndrome in a patient        comprising:    -   a) measuring a level of IL-6 in a urine sample or a plasma        sample from the patient,    -   b) comparing the measured level of IL-6 to a predetermined        reference level,    -   c) determining whether or not the measured level of IL-6 is        greater than the corresponding reference level    -   wherein when the measured level of IL-6 is greater than the        corresponding reference level, treatment for cardiorenal        syndrome is administered.        65. The method of embodiment 64, wherein the patient is a heart        failure patient.        66. The method of embodiment 64 or embodiment 65, wherein the        level of IL-6 is measured using an enzyme-linked immunosorbent        assay (ELISA).        67. The method of any one of embodiments 64-66, wherein the        treatment administered for cardiorenal syndrome comprises        administering one or more of at least one diuretic,        angiotensin-converting-enzyme inhibitor, angiotensin receptor        blocker, natriuretic peptide, adenosine antagonist, an IL-6        antagonist, or any combination thereof.        68. The method of embodiment 67, wherein treating cardiorenal        syndrome comprises administering at least one anti-IL-6 antibody        or anti-IL-6R antibody.        69. A kit comprising reagents for an assay for measuring a level        of IL-6 and written instructions, the written instructions        comprising:    -   a) measuring a level of IL-6 in a urine or plasma sample from        the subject,    -   b) comparing the measured level of IL-6 to a predetermined        reference level,    -   c) determining whether or not the measured level of IL-6 is        greater than the corresponding reference level,    -   wherein when the measured level of IL-6 is greater than the        corresponding reference level, the subject has cardiorenal        syndrome, and treatment is recommended.        70. The kit of embodiment 69, wherein the instructions direct        the kit for use on a heart failure patient.        71. The kit of embodiment 69 or 70, wherein the kit contains        reagents for measuring the level of IL 6 using an enzyme-linked        immunosorbent assay (ELISA).

8.8. Experimental Examples

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, practice the claimed methods of the presentinvention. The following working examples therefore, specifically pointout the preferred embodiments of the present invention, and are not tobe construed as limiting in any way the remainder of the disclosure.

The materials and methods employed in the experiments disclosed hereinare now described.

8.8.1. Example 1: Urine and Plasma IL-6 Associations in Heart FailurePatients

Methods.

Consecutive heart failure (“HF”) patients receiving high dose diuretictherapy at the Yale Transitional Care Clinic (YTCC) were enrolled in theprospective observational study.

The YTCC is an outpatient clinic with a focus on diuretic and fluidstatus management. Patients present for 4-8 hours of treatment, duringwhich they receive 1-3 doses of loop diuretic. The dosing protocol isdetermined by patient fluid status; the choice of oral (PO) torsemide orintravenous (IV) bumetanide is at the discretion of the treatingphysician. During the treatment period all urine produced is saved in acumulative collection container and sent to the clinical laboratory forelectrolyte measurements at the conclusion of the visit. A cumulativeurine collection is conducted during the treatment period. Additionalspot urine samples are saved both before and one hour after diureticadministration.

Assays.

A Randox RxDaytona™ automated clinical chemistry analyzer was used tomeasure urine and serum electrolytes using ion selective electrodes.Urea, creatinine, bicarbonate and cystatin C were measured using Randoxreagents per the manufacturer's instructions (Randox Laboratories™, UK).Concentrations of interleukin 10 and IL-6 were measured using theMesoScale Discovery (MSD) platform (Meso Scale Diagnostics™Gaithersburg, Md., USA). Levels of amino terminal pro B-type natriureticpeptide (NT-proBNP) were measured at the Yale clinical laboratory on aRoche Elecsys 120 analyzer (Roche Diagnostics, Indianapolis, USA).Plasma renin activity (PRA), angiotensinogen and active renin weremeasured using commercially available competitive ELISA kits from ALPCO™per manufacturer's instructions (ALPCO™, Salem, N.H., USA). Total reninwas also analyzed using commercially available ELISA kits (R&D Systems™,Minneapolis, USA). The total renin immunoassay kit from R&D Systems™recognizes both active renin and prorenin. The assay's mean detectablelimit is 4.43 pg/ml for total renin and 0.81 pg/ml for active renin.Liquid chromatography mass spectrometry was used to measure levels ofbumetanide and torsemide in urine. Ultra-high performance liquidchromatography was performed on the Agilent Infinity 1290 UPLC System™.Chromatographic separation was achieved on the Zorbax Bonus RP™ 2.1×50mm 1.8μ column with a flow rate of 0.6 ml/min. The mobile phasecontained of 0.1% Formic acid (Buffer A) and 80% acetonitrile in 0.1%Formic acid (Buffer B). Mass spectrometry was performed on Agilent Q-TOFSystem™ (Agilent™, Santa Clara, Calif., USA) in positive ion mode.

Calculations and Definitions.

eGFR was calculated using the Chronic Kidney Disease EpidemiologyCollaboration (CKD-EPI) formula. Doses of loop diuretics were convertedto furosemide equivalents with 1 mg bumetanide=20 mg torsemide=40 mgintravenous furosemide=80 mg oral furosemide. As published previously,diuretic efficiency was defined as the increase in urinary sodium outputover the treatment period per each doubling of the loop diuretic dose,centered on 40 mg of furosemide equivalents; this scale was chosen toaccount for the sigmoidal dose-response curve of these drugs. Urinarydiuretic excretion was calculated by multiplying the concentration ofdiuretic in the urine by the volume of urine produced in the first 3hours after diuretic administration; this quantity of estimated diureticwas then normalized to the administered diuretic dose in furosemideequivalents, taking into account the published urinary clearance of theparticular diuretic received by the patient (bumetanide or torsemide).Fractional excretions of sodium and potassium were calculated using theformula: Fractional excretion of X (FEX)=[X]Urine*[Creatinine]Serum/([X]Serum or plasma*[Creatinine]Urine). Urine proteins including renin,angiotensinogen and IL-6 were indexed to urinary creatinine. A low urineIL-6 was defined as a value less than the median of 14.2 pg/g of urinarycreatinine. A low plasma IL-6 was defined as a value less than themedian of 2.0 pg/mL. High levels of urine and plasma renin andangiotensinogen were defined as values greater than or equal to thecohort medians of these variables.

Statistical Analysis.

Values reported are mean+/−SD, median (quartile 1-quartile 3) andpercentage. Correlations between continuous variables are Spearman's rhowith the exception of adjusted correlations. Pearson's chi-squared testwas used to compare categorical variables between groups. To comparecontinuous variables between groups, either Student's t-test or theWilcoxon Rank Sum test was used. A log transformation was applied toskewed variables including plasma IL-6, urine IL-6 and NT-pro-BNP beforeentering them into multivariable models and partial correlationanalyses. Logistic regression was used to evaluate association betweenthe odds of low diuretic efficiency, an eGFR<60 mL/min/1.73 m², or highlevels of urine or plasma neurohormonal parameters with plasma and urinelevels of IL-6, both on a univariate level and with adjustment forplasma or urine IL-6 and/or eGFR. Cox proportional hazards modeling wasused to evaluate time-to-event associations with all-cause mortality.Statistical analysis was performed with IBM SPSS Statistics version 23(IBM Corp., Armonk, N.Y.) and Stata version 13 (StataCorp™, CollegeStation, Tex.). Statistical significance was defined as 2-tailed p<0.05for all analyses.

The baseline characteristics of our population are described in Table 2,below. In this subset, 98 patients underwent determination of IL-6levels in blood and urine. The median (IQR) pre-diuretic level of urineIL-6 was 14.2 pg/g of creatinine (5.6-36.2 pg/g) whereas the medianlevel of plasma IL-6 was 2.0 pg/mL (1.2-3.9 pg/mL). Plasma and urineIL-6 levels were only modestly correlated (r=0.40, p<0.001). Notably,those with lower than median levels of urine IL-6 tended to be younger,Caucasian, more often on angiotensin receptor blocker (ARB) orangiotensin converting enzyme (ACE-I) therapy, more likely to have heartfailure with reduced ejection fraction (HFrEF), a higher GFR andsubstantially lower plasma levels of NT-proBNP levels. The profile ofpatients defined by a lower than median plasma IL-6 was somewhatdifferent, but largely reflected the same trends.

TABLE 2 Baseline Characteristics Overall Cohort Low Urine IL-6 HighUrine IL-6 (n = 98) (n = 49) (n = 49) Demographics Age 67.9 ± 13.5 62.1± 13.2 73.8 ± 11.2 Black race, % 36 57 15 Male sex, % 44 49 39 PastMedical History, % Hypertension 91 94 88 Diabetes 47 55 39 Gout 13 10 16Ischemic etiology 30 33 27 Post-discharge follow-up visit 63 71 55Baseline Medications, % ACEI or ARB 55 65 45 Beta-blocker 77 82 71Thiazide-type diuretic 14 12 16 MRA 33 37 29 Digoxin 10 12  8 Home loopdiuretic dose (mg 80 (60-160) 80 (60-160) 100 (80-160) furosemideequivalents) Physical Exam Weight (kg) 98.2 ± 33.4 106.0 ± 37.9  90.4 ±26.4 BMI 34.8 ± 10.8 36.7 ± 11.3 32.5 ± 9.9  Systolic blood pressure(mmHg) 122.6 ± 18.9  120.6 ± 19.1  124.5 ± 18.7  Heart rate (beats perminute): 75.9 ± 13.2 75.6 ± 13.2 76.2 ± 13.4 Echocardiogram Leftventricular ejection fraction 45 (27-56) 35 (25-50) 51 (31-59) (%)Ejection fraction ≥40% 53% 43% 63% Laboratory Values Sodium (mmol/L) 136(134-140) 137 (134-138) 139 (134-141) Chloride (mmol/L) 96 (95-102) 100(96-102) 98 (95-102) Hypochloremia, % 34 27 42 Potassium (mmol/L) 4.1(3.7-4.5) 4.2 (3.8-4.6) 4.1 (3.6-4.3) Bicarbonate (mmol/L) 23.9(21.7-26.4) 23.1 (21.6-25.3) 24.9 (22.2-27.9) Blood urea nitrogen(mg/dL) 29.0 (20.0-47.0) 27.0 (17.5-44.0) 32.0 (24.0-48.0) Serumcreatinine (mg/dL) 1.5 ± 0.8 1.4 ± 0.6 1.7 ± 0.9 BUN/Creatinine ratio24.0 ± 8.3  24.1 ± 8.1  23.9 ± 8.5  Estimated glomerular filtration 54.9± 28.2 64.3 ± 30.1 45.7 ± 22.8 rate (eGFR) (mL/min/1.73 m²) eGFR <60mL/min/1.73 m² 61% 48% 73% Albumin (g/dL) 3.8 ± 0.4 3.9 ± 0.4 3.8 ± 0.4Hemoglobin (g/dL) 12.2 ± 2.3  12.7 ± 2.2  11.7 ± 2.3  DiureticParameters Diuretic dose administered 140 (40-240) 80 (40-160) 160(80-280) (mg furosemide equivalents) Diuretic administered, % IVBumetanide: 45 27 63 Torsemide: 55 73 37 Low Plasma IL-6 High PlasmaIL-6 p-value (n = 49) (n = 49) p-value Demographics Age <0.001* 65.8 ±15.1 70.1 ± 11.5 0.12 Black race, % <0.001* 47 26 0.04* Male sex, % 0.3149 39 0.31 Past Medical History, % Hypertension 0.29 96 86 0.08 Diabetes0.11 37 57 0.04* Gout 0.37 10 16 0.37 Ischemic etiology 0.46 33 27 0.46Post-discharge follow-up visit 0.09 61 65 0.68 Baseline Medications, %ACEI or ARB 0.04* 67 43 0.02* Beta-blocker 0.23 82 71 0.23 Thiazide-typediuretic 0.56  6 22 0.02* MRA 0.39 31 35 0.67 Digoxin 0.50  8 12 0.50Home loop diuretic dose (mg 0.60 80 (40-120) 160 (80-200) 0.002*furosemide equivalents) Physical Exam Weight (kg) 0.02* 98.4 ± 31.5 98.0± 35.5 0.95 BMI 0.09 33.5 ± 8.4  36.2 ± 13.0 0.28 Systolic bloodpressure (mmHg) 0.32 123.4 ± 18.9  121.7 ± 19.1  0.65 Heart rate (beatsper minute): 0.82 74.9 ± 13.3 76.8 ± 13.2 0.48 Echocardiogram Leftventricular ejection fraction 0.03* 39 (29-54) 45 (26-57) 0.56 (%)Ejection fraction ≥ 40% 0.04* 49% 57% 0.42 Laboratory Values Sodium(mmol/L) 0.06 138 (135-140) 136 (132-140.0) 0.12 Chloride (mmol/L) 0.50101 (98-103) 96 (91-101) <0.001* Hypochloremia, % 0.13 14 55 <0.001*Potassium (mmol/L) 0.053 4.1 (3.7-4.5) 4.1 (3.7-4.4) 0.64 Bicarbonate(mmol/L) 0.03* 23.1 (21.5-25.2) 25.0 (22.6-28.0) 0.03* Blood ureanitrogen (mg/dL) 0.10 27.0 (19.0-39.0) 36.0 (23.0-65.0) 0.04* Serumcreatinine (mg/dL) 0.046* 1.3 ± 0.5 1.8 ± 1.0 0.007* BUN/Creatinineratio 0.92 23.6 ± 8.0  24.5 ± 8.5  0.58 Estimated glomerular filtration<0.001* 61.4 ± 27.2 48.3 ± 27.8 0.02* rate (eGFR) (mL/min/1.73 m²) eGFR<60 mL/min/1.73 m² 0.01* 55% 67% 0.24 Albumin (g/dL) 0.045* 4.0 ± 0.43.7 ± 0.4 <0.001* Hemoglobin (g/dL) 0.04* 12.8 ± 2.0  11.5 ± 2.4  0.004*Diuretic Parameters Diuretic dose administered 0.001* 80 (40-160) 160(80-320) <0.001* (mg furosemide equivalents) Diuretic administered, % IVBumetanide: <0.001* 33 57 0.02* Torsemide: 67 43 ACE-I =angiotensin-converting enzyme inhibitor. ARB = angiotensin receptorblocker. MRA = mineralocorticoid receptor antagonist. IL = interleukin.BUN = blood urea nitrogen. BNP = brain natriuretic peptide. BMI = bodymass index. *p < 0.05.

TABLE 3 Plasma and Urine Biomarkers at Baseline Overall Cohort Low UrineIL-6 High Urine IL-6 (n = 98) (n = 49) (n = 49) Plasma Biomarkers IL-6(pg/mL) 2.0 (1.2-3.9) 1.4 (0.9-2.9) 2.8 (1.5-6.1) IL-10 (pg/mL) 0.3(0.2-0.7) 0.3 (0.2-0.4) 0.5 (0.3-0.8) IL-10/IL-6 ratio 0.2 (0.1-0.4) 0.2(0.1-0.3) 0.2 (0.1-0.4) Total renin (pg/mL) 1167.7 (734.7-2759.7) 1169.2(734.7-3013.5) 1166.3 (816.0-2390.9) Active renin (pg/mL) 36.8(8.4-162.8) 79.0 (7.7-184.2) 28.1 (12.0-139.4) Angiotensinogen (ng/mL)167.9 (54.1, 445.3) 193.9 (48.2, 518.1) 160.3 (58.3, 430.2) NT pro-BNP(pg/mL) 1825.0 (681.0-5030.0) 840.0 (256.0-3115.0) 3115.0(1440.0-6070.0) Urine Biomarkers IL-6 (pg/g of creatinine) 14.2(5.6-36.2) 5.6 (2.5-7.7) 36.2 (20.7-68.9) IL-6 (pg/mL) 1.2 (0.5-2.6) 0.5(0.2-0.7) 2.5 (1.6-4.0) Renin (pg/mg of 0.3 (0.1-0.8) 0.2 (0.1-0.5) 0.6(0.3-1.2) creatinine) Renin (pg/mL) 28.2 (10.2-88.3) 19.9 (7.5-53.1)37.0 (13.4-109.6) Angiotensinogen (pg/mg 18.7 (5.4-126.3) 6.2 (3.3-19.0)123.5 (17.3-549.7) creatinine) Angiotensinogen (pg/mL) 1530 (624-10941)966 (231-1567) 5850 (1518-53865) FE_(Na) (%) 0.5 (0.2-1.3) 0.4 (0.2-1.2)0.5 (0.2-1.4) FE_(K) (%) 18.4 (11.7-39.3) 15.5 (9.9-32.5) 21.6(15.2-44.6) FE_(Urea) (%) 63.6 (44.8-81.3) 59.5 (44.4-80.5) 65.5(44.9-81.6) Low Plasma IL-6 High Plasma IL-6 p-value (n = 49) (n = 49)p-value Plasma Biomarkers IL-6 (pg/mL) <0.001* 1.2 (0.9-1.4) 3.9(2.9-6.8) <0.001* IL-10 (pg/mL) 0.001* 0.3 (0.2-0.5) 0.5 (0.3-0.8) 0.01*IL-10/IL-6 ratio 0.73 0.3 (0.2-0.6) 0.1 (0.1-0.2) <0.001* Total renin(pg/mL) 0.97 909.6 (595.2-1517.9) 1784.4 (921.6-3868.7) <0.001* Activerenin (pg/mL) 0.38 13.0 (4.4-160.7) 49.1 (15.7-169.2) 0.07Angiotensinogen (ng/mL) 0.81 178.8 (31.6, 481.2) 167.2 (75.5, 445.3)0.50 NT pro-BNP (pg/mL) <0.001* 1440.0 (412.0-3010.0) 3390.0(792.0-6410.0) 0.01* Urine Biomarkers IL-6 (pg/g of creatinine) <0.001*7.0 (3.2-19.1) 21.2 (9.2-45.3) <0.001* IL-6 (pg/mL) <0.001* 0.8(0.4-1.9) 1.7 (0.6-3.2) 0.03* Renin (pg/mg of <0.001* 0.2 (0.1-0.6) 0.5(0.2-1.5) <0.001* creatinine) Renin (pg/mL) 0.01* 23.7 (7.5-53.1) 35.9(12.3-109.6) 0.06 Angiotensinogen (pg/mg <0.001* 7.3 (3.7-39.7) 33.7(12.3-318.9) 0.002* creatinine) Angiotensinogen (pg/mL) <0.001* 1105(463-5801) 2153 (966-23952) 0.02* FE_(Na) (%) 0.22 0.4 (0.1-1.0) 0.7(0.3-1.8) 0.02* FE_(K) (%) 0.02* 15.8 (9.6-24.5) 24.5 (13.9-50.3) 0.001*FE_(Urea) (%) 0.62 66.7 (47.5-82.8) 58.2 (44.6-76.0) 0.20 IL =interleukin. NT pro-BNP = N-terminal pro b-type natriuretic peptide.FENa = fractional excretion of sodium. FEK = fractional excretion ofpotassium. FEUrea = fractional excretion of urea.

8.8.1.1. Kidney Function and IL-6

As shown in Table 2, eGFR was lower in patients with high urine orplasma IL-6, but this association was only significant between eGFR andurine IL-6 (p=0.01). A correlation between both plasma IL-6 and eGFR(r=−0.26, p=0.01) as well as urine IL-6 and eGFR (r=−0.38, p<0.001) wasobserved. However, on adjustment for urine IL-6, there was no longer asignificant association between plasma IL-6 and eGFR (p=0.20), whereas asignificant association remained between urine IL-6 and eGFR afteradjustment for plasma IL-6 (partial r=−0.32, p=0.002). Similarly, therisk of reduced eGFR as defined by an eGFR<60 mL/min/1.73 m² wasincreased with higher levels of urine IL-6 (OR=1.9 per SD increase, 95%CI=1.2, 3.1, p=0.006) and not with higher levels of plasma IL-6 (OR=1.3per SD increase, 95% CI=0.8-2.0, p=0.25).

8.8.1.2. Diuretic Response and IL-6

There was an inverse association between diuretic efficiency and bothurine IL-6 (r=−0.43, p<0.001) and plasma IL-6 (r=−0.31, p=0.002; FIG.3). Odds for a low diuretic efficiency increased with higher levels ofeither urine IL-6 (OR=2.3 per SD increase, 95% CI 1.4-3.8, p=0.001) orplasma IL-6 (OR=1.7 per SD increase, 95% CI=1.1-2.7, p=0.02). Uponadjustment for eGFR, only urine IL-6 remained significantly associatedwith risk of low diuretic efficiency (adjusted OR=1.8 per SD increase,95% CI 1.1-3.1, p=0.02; FIG. 3). Furthermore, when urine IL-6 and plasmaIL-6 were both entered into a logistic regression model, only urine IL-6remained associated with risk of low diuretic efficiency (adjustedOR=2.1 per SD increase, 95% CI 1.3-3.5, p=0.004) while plasma IL-6showed no such association (OR=1.4, 95% CI 0.9-2.2, p=0.17).

8.8.1.3. Neurohormonal Activation

Plasma IL-6 was associated with higher levels of plasma renin (Table 3and FIG. 3). Notably, higher levels of plasma IL-6 conferred additionalrisk of high plasma total renin (OR=1.9 per SD increase, 95% CI 1.2-3.0,p=0.008), and this persisted despite adjustment for use of ACE-I or ARBand urine IL-6 levels (adjusted OR=2.3 per SD increase, 95% CI 1.3-3.9,p=0.003). Urine IL-6 was not associated with increased risk of highplasma renin (OR=1.0, 95% CI 0.7-1.5, p=0.98).

Urine IL-6 was strongly associated with high levels of tissue level RAASactivation as measured by urine angiotensinogen (OR=4.2 per SD increase,95% CI 2.2-7.9, p<0.001) and urine renin (OR=2.1 per SD increase, 95% CI1.3-3.4, p=0.002; FIG. 3). These associations persisted after adjustmentfor plasma IL-6 levels and ACE-I or ARB use (adjusted OR for high urineangiotensinogen=4.2 per SD increase, 95% CI 2.2-8.3, p<0.001; adjustedOR for high urine renin=2.0 per SD increase, 95% CI 1.2-3.3, p=0.005).Plasma IL-6 did not demonstrate a univariate association with risk ofhigh urine renin (OR=1.3 per SD increase, 95% CI 0.9-2.0, p=0.16).Although a trend toward association with urine angiotensinogen (OR=1.5per SD increase, 95% CI 0.98-2.3, p=0.06) was observed, it waseliminated with adjustment for urine IL-6 (adjusted OR=1.1, 95% CI0.7-1.7, p=0.76).

8.8.1.4. Association with Survival

Over a median follow-up time of 713 days, 32 deaths occurred. Consistentwith previous reports, increases in plasma IL-6 were associated with ahigher risk of mortality (univariate HR=2.8 per SD increase, 95% CI2.0-4.0, p<0.001). Plasma IL-6 remained associated with mortality aftermultivariable adjustment for baseline characteristics including age,race, baseline NT-proBNP levels, use of ACE-I or ARB, home loop diureticdose and eGFR (adjusted HR=2.3 per SD increase, 95% CI=1.5-3.7,p<0.001). In contrast, urine IL-6 was not associated the risk ofmortality (univariate HR=1.3 per SD increase, 95% CI 0.9-1.8, p=0.15;adjusted HR=1.02, 95% CI=0.6-1.6, p=0.93).

8.8.1.5. Summary

Plasma and urine IL-6 levels capture distinctive aspects of the role ofIL-6 in cardiorenal disease pathophysiology. Plasma IL-6 levels areassociated with global measures of disease severity such as risk ofmortality. In contrast, urine IL-6, likely a measure of kidneyinflammation, is strongly correlated with multiple measures ofcardiorenal syndrome (“CRS”) in these heart failure patients, includingdiuretic resistance, renin angiotensin aldosterone and system (RAAS)activation, and lower estimated glomerular filtration rate (eGFR).

These data demonstrate that urine IL-6 level is a useful biomarker forrenal inflammation, and is particularly useful in assessing renalsymptoms in heart failure patients. In addition, serial urine IL-6levels should prove useful in monitoring and assessing the renal benefitof therapeutic interventions in heart failure.

8.8.2. Example 2: TMPRSS6 Genotype Stratifies Heart Failure PatientsWhose Renal Symptoms are Predicted to be Responsive to IL-6 Antagonists

The data obtained in Example 1 also predict that treatment with an IL-6antagonist should be effective in reducing renal inflammation in heartfailure patients.

However, because infection is often a precipitating cause of acutedecompensation in heart failure patients, it is important to limitanti-cytokine and other immunosuppressive therapies to those heartfailure patients who are likely to respond with improved renal and/orcardiac function. The cost of chronic IL-6 antagonist therapy alsomilitates for limiting treatment to those heart failure patients who arelikely to respond with improved renal and/or cardiac function.

Analysis conducted in Example 1 was expanded to 129 patients. FIG. 1A isa bar graph showing diuretic efficiency (“DE”) by tertiles of urineIL-6, confirming the inverse correlation of urinary IL-6 with diureticefficiency observed in the 98 patient subset. FIG. 1B is a bar graphshowing diuretic efficiency (“DE”) by tertiles of plasma IL-6 in these129 patients, confirming an inverse correlation of plasma IL-6 withdiuretic efficiency (uncorrected by urinary IL-6 levels).

Each patient's genotype at the rs855791 single nucleotide polymorphism(“SNP”) in transmembrane protease serine 6 (“TMPRSS6”) was furtherassessed. The TMPRSS6 polypeptide, also known as Matriptase-2 (MT2),cleaves hemojuvelin and inhibits bone morphogenetic protein signaling.The rs855791 (G2321A) SNP alters the TMPRSS6 protein sequence: theallele with highest frequency in the human population (the major allele)is 2321G, encoding 736A; the allele with lowest frequency in the humanpopulation (minor allele) is 2321A, encoding 736V.

Genomic DNA was isolated from buffy coats using ReliaPrep large volumeHT gDNA isolation system on the HSM Instrument (Promega, Madison, USA).The purity of the isolated DNA was assessed by Nanodrop. Genotyping wascarried out at the Yale Centre for Genome analysis. Whole genomegenotyping was done using Infinium® Exome-24 v1.0 BeadChip Kit fromIllumina using standard protocols suggested by the manufacturer(Illumina, Inc., San Diego, Calif.). The amplification, fragmentation,precipitation, resuspension and hybridization steps were done manually.The arrays were scanned on the Illumina Hiscan instrument. The IlluminaHiScan or iScan System scans the BeadChip, using a laser to excite thefluorophore of the single-base extension product on the beads. Thescanner records high resolution images of the light emitted from thefluorophores. The Illumina GenomeStudio Genotyping Module, included withthe Illumina Infinium Assay system, was used for extracting genotypingdata from intensity data files (*.idat files) collected from theIllumina HiScan System. The Infinium Exome-24 v1.0 BeadChip containsover 240,000 putative functional exonic variants selected from over12,000 individual human exome and whole-genome sequences. The >240,000markers represent diverse populations, including European, African,Chinese, and Hispanic individuals, and a range of common conditions,such as type 2 diabetes, cancer, metabolic, and psychiatric disorders.Detailed Illumina genotyping protocol is available atsupport.illumina.com (infinium_hts_assay_protocol_user_guide_15045738_a.pdf). The details of the SNPs in this exome chip are available atsupport.illumina.com/downloads/infinium-exome-24-v1-O-product-files.html.

As shown in FIG. 2A, urine levels of IL-6 were inversely correlated withdiuretic efficiency only in the patients having at least one copy of themajor allele of the TMPRSS6 rs855791 SNP (FIG. 2A, right panel,“AG+GG”); urine levels of IL-6 are not significantly correlated withdiuretic efficiency in patients homozygous for the minor allele (FIG.2A, left panel, “AA”).

As shown in FIG. 2B, plasma levels of IL-6 correlated inversely withdiuretic efficiency only in the patients having at least one copy of themajor allele of the TMPRSS6 rs855791 SNP (FIG. 2B, right panel,“AG+GG”); plasma levels of IL-6 are not significantly correlated withdiuretic efficiency in patients homozygous for the minor allele (FIG.2B, left panel, “AA”).

These data indicate that treatment of heart failure with an IL-6antagonist will only improve renal symptoms in heart failure patientshaving at least one copy of the TMPRSS6 rs855791 major allele.

8.8.3. Example 3: Correlation of IL-6 with the Expression of NKCC2,ENaC-Beta, and NCC in the Absence or Presence of Ruxolitinib

Methods

The mouse M1 CCD cell line (American Type Culture Collection (ATCC), Cat#CRL-2038) was maintained the cell culture medium containing equalamount of DMEM (Sigma-Aldrich, Cat #D6046) and Han F12 (Sigma-Aldrich,Cat #11765-047), supplemented with 5% Fetal Bovine Serum (FBS), 1%Penicillin-Streptomycin (Thermo Fisher Scientific, Cat #15140-122), 1%Insulin-Transferrin-Selenium (Thermo Fisher Scientific, Cat #51500-056)and 100 nM Dexamethasone (Sigma-Aldrich, Cat #D4902-100MG).

M1 CCD cells were seeded at 1 million/well in 6-well plate in the cellculture medium, and incubated overnight in a 37° C., 5% CO₂ incubator onDay 0. The cell culture medium was changed to DMEM/F12 serum-free mediumon Day 1 and the cells were incubated overnight in a 37° C., 5% CO₂incubator. On Day 2, the serum-free medium was removed and the cellculture medium was added to each well. Ruxolitinib (Selleckchem, Cat#51378) was added at the final concentration of 1 μM or 100 μM 10 minbefore the addition of IL-6 (Sigma-Aldrich, Cat #SRP3096-20UG) at thefinal concentration of 10 ng/mL, 100 ng/mL, or 500 ng/mL. A control wellwithout Ruxolitinib or IL-6 was included. The cells were treated for 24hours with IL-6 and/or Ruxolitinib. The cells of each well were washedwith once with 1×PBS and collected in 250 μL 1×PIPA buffer (10×,Millipore, Cat #20-188) supplemented with 1% protease inhibitor cocktail(100×, Thermo Fisher Scientific, Cat #78430). The sample were analyzedby immunoblotting using anti-NKCC2 antibody (Millipore, Cat #AB3562P),anti-ENaC-beta antibody (Millipore, Cat #AB3532P), or anti-NCC antibody(Millipore, Cat #AB3553) and the protein expression was quantified. Eachexperiment was done in triplicate.

Results

We examined the expression of NKCC2 (Na—K—Cl cotransporter 2), ENaC-beta(epithelial sodium channel, beta subunit), and NCC (sodium-chloridesymporter) proteins in mouse M1 CCD cell line after treatment of IL-6 inthe absence or presence of a JAK inhibitor, Ruxolitinib. The mouse M1CCD cells are genotypically analogous to human cells homozygous for theTMPRSS6 rs855791 major allele. As shown in FIGS. 4A, 4B, and 4C, thetreatment of IL-6 increased the expression of NKCC2, ENaC-beta, and NCC.Ruxolitinib blocked the effect of IL-6 on the ion transporter proteins.

Increased expression of these ion transporters provides a putativemechanism for IL-6 mediated diuretic resistance.

Because the increased expression is not known to be linked to hepcidinexpression, these data suggested that IL-6 antagonism could be effectivein treating diuretic resistance even in patients homozygous for theTMPRSS6 rs855791 minor allele.

8.8.4. Example 4: Correlation of IL-6 with the Expression of NKCC2,ENaC-Beta, and NCC in the Absence or Presence of Spironolactone

Methods

The mouse M1 CCD cell line (American Type Culture Collection (ATCC), Cat#CRL-2038) was maintained the cell culture medium containing equalamount of DMEM (Sigma-Aldrich, Cat #D6046) and Han F12 (Sigma-Aldrich,Cat #11765-047), supplemented with 5% Fetal Bovine Serum (FBS), 1%Penicillin-Streptomycin (Thermo Fisher Scientific, Cat #15140-122), 1%Insulin-Transferrin-Selenium (Thermo Fisher Scientific, Cat #51500-056)and 100 nM Dexamethasone (Sigma-Aldrich, Cat #D4902-100MG).

M1 CCD cells were seeded at 1 million/well in 6-well plate in the cellculture medium, and incubated overnight in a 37° C., 5% CO₂ incubator onDay 0. The cell culture medium was changed to DMEM/F12 serum-free mediumon Day 1 and the cells were incubated overnight in a 37° C., 5% CO₂incubator. On Day 2, the serum-free medium was removed and the cellculture medium was added to each well. Spironolactone (Selleckchem, Cat#54054) was added at the final concentration of 1 μM or 100 μM 10 minbefore the addition of IL-6 (Sigma-Aldrich, Cat #SRP3096-20UG) at thefinal concentration of 10 ng/mL, 100 ng/mL, or 500 ng/mL. A control wellwithout Spironolactone or IL-6 was included. The cells were treated for24 hours with IL-6 and/or Spironolactone. The cells of each well werewashed with once with 1×PBS and collected in 250 μL 1×PIPA buffer (10×,Millipore, Cat #20-188) supplemented with 1% protease inhibitor cocktail(100×, Thermo Fisher Scientific, Cat #78430). The sample were analyzedby immunoblotting using anti-NKCC2 antibody (Millipore, Cat #AB3562P),anti-ENaC-beta antibody (Millipore, Cat #AB3532P), or anti-NCC antibody(Millipore, Cat #AB3553) and the protein expression was quantified. Eachexperiment was done in triplicate.

Results

We examined the expression of NKCC2 (Na—K—Cl cotransporter 2), ENaC-beta(epithelial sodium channel, beta subunit), and NCC (sodium-chloridesymporter) proteins in mouse M1 CCD cell line after treatment of IL-6 inthe absence or presence of a potassium-sparing diuretic, Spironolactone.The mouse M1 CCD cells are genotypically analogous to human cellshomozygous for the TMPRSS6 rs855791 major allele. As shown in FIGS. 5A,5B, and 5C, the treatment of IL-6 increased the expression of NKCC2,ENaC-beta, and NCC. Spironolactone blocked the effect of IL-6 on the iontransporter proteins.

Increased expression of these ion transporters provides a putativemechanism for IL-6 mediated diuretic resistance.

Because the increased expression is not known to be linked to hepcidinexpression, these data suggested that IL-6 antagonism could be effectivein treating diuretic resistance even in patients homozygous for theTMPRSS6 rs855791 minor allele.

8.8.5. Example 5: Association of IL-6 with Diuretic Response in PatientsHospitalized for Acute Heart Failure (HF)

Methods.

Data from the PROTECT trial (Weatherley et al., 2010, J. Card. Fail.16:25-35; Massie et al., 2010, N Engl. J. Med. 363:1419-1428) wasanalyzed according to tertiles of IL-6. The PROTECT trial was arandomized placebo-controlled trial testing the effect of AdenosineA1-Receptor Antagonist Rolofylline on dyspnea relief, risk of worseningrenal function and clinical outcomes. The key inclusion and exclusioncriteria of the trial are shown below.

TABLE A Inclusion Exclusion >18 years Acute contrast-induced nephropathyHistory of HF >14 days with diuretic therapy Temp >38 or sepsisrequiring IV Hospitalized for ADHF* requiring IV diuretic antimicrobialtreatment therapy Serum potassium <3.5 mEq/L Within 24 hrs ofpresentation to the hospital Ongoing or planned IV therapy for ADHFAnticipated need for IV furosemide >40 mg/day for with positiveinotropic agents, vasopressors, at least 24 hr vasodilators Impairedrenal function defined as creatinine BNP <500 pg/mL or NT-pro-BNP <2000clearance of admission between 20 and 80 mL/min pg/mL (Cockcroft-Gault)Ongoing or planned treatment with Systolic blood pressure >95 mm Hgultrafiltration Severe pulmonary disease Clinical evidence of acutecoronary syndrome in the 2 weeks before screening Hgb <8 g/dL, or Hct<25%, or the need for a blood transfusion Systolic blood pressure $160mm Hg at randomization *ADHF: dyspnea at rest or with minimal exertionand signs of fluid overload manifested by at least one of the followingat time of randomization: JVP >8 cm, or Pulmonary rales ≥1/3 up the lungfields, not clearing with cough, or ≥2 

 peripheral edema, or presacral edema

In total, 2033 patients with ADHF were included in the PROTECT study. Ofthese patients, IL-6 was measured by Singulex in 1445 patients atadmission (baseline), 1462 patients at day 2 (24 hr after baseline) and1445 patients at day 7. Diuretic response was defined as weight changeon day 4 per 40 mg of furosemide (or equivalent doses) administered frombaseline to day 3. The primary endpoint of this study was all-causemortality at 180 days.

Statistical Analysis.

Baseline characteristics are presented according to tertiles of IL-6.Differences between tertiles of baseline characteristics were testedusing one-way analysis of variance (ANOVA), Kruskal Wallis or chi2-testwhere appropriate. Univariable linear regression was performed usingdiuretic response as the dependent variable and (log-transformed) IL-6at baseline as the independent variable correcting for clinicallyrelevant variables associated with diuretic response. Survival analysiswas performed using Cox regression analysis correcting for clinicallyrelevant variables and the PROTECT risk model (O'Connor et al., 2012,Eur. J. Heart Fail. 14:605-612). The PROTECT risk model includes:previous hospitalization for HF, edema, systolic blood pressure, sodiumlevels, BUN, creatinine and albumin at admission.

Results.

The baseline characteristics of the population are described in Table 4,below. Higher levels of IL-6 at baseline are associated with higherlevels of BNP, anemia, eGFR<60 and older age (FIG. 6).

TABLE 4 Baseline Characteristics Factor 1st tertile 2nd tertile 3rdtertile 531 530 530 N 0.66-7.8 pg/mL 7.9-16.1 pg/mL 16.2-274.2 pg/mLp-value Demographics Age 69.2 (11.5) 70.8 (11.0) 72.5 (10.8) <0.001 Sex347 (65.3%) 349 (65.8%) 354 (66.8%) 0.88 BMI 28.3 (5.8) 28.8 (6.0) 29.1(6.2) 0.093 eGFR 52.5 (20.2) 47.2 (19.3) 44.9 (17.4) <0.001 NYHA classI/II 107 (21.0%) 71 (14.1%) 96 (19.2%) 0.008 III 267 (52.4%) 263 (52.2%)237 (47.4%) IV 136 (26.7%) 170 (33.7%) 167 (33.4%) LVEF 30 (25, 40) 30(20, 40) 30 (22, 40) 0.35 HFpEF 42 (15.6%) 39 (16.3%) 38 (14.9%) 0.92Systolic BP 126.2 (17.4) 124.5 (17.5) 123.6 (17.4) 0.043 Diastolic BP75.3 (11.2) 73.5 (12.1) 72.3 (11.9) <0.001 Heart Rate 79.2 (14.6) 79.4(15.8) 81.3 (16.0) 0.052 Respiratory rate 20.6 (4.3) 21.2 (4.1) 21.7(4.9) <0.001 Medical history Atrial fibrillation 95 (45.5%) 92 (43.6%)78 (39.4%) 0.45 Valve disease 196 (37.0%) 195 (36.8%) 208 (39.5%) 0.61Mitral regurgitation 174 (32.8%) 178 (33.6%) 184 (34.8%) 0.78 Aorticstenosis 18 (3.4%) 16 (3.0%) 32 (6.0%) 0.027 Aortic regurgitation 43(8.1%) 18 (3.4%) 38 (7.2%) 0.004 Heart failure (HF) 510 (96.0%) 505(95.3%) 501 (94.5%) 0.51 Hospitalization for HF 258 (48.6%) 266 (50.2%)259 (48.9%) 0.86 previous year No. of HF hospitalizations, 1.0 (1.0,2.0) 1.0 (1.0, 2.0) 1.0 (1.0, 2.0) 0.62 median (IQR) Ischemic heartdisease 362 (68.4%) 396 (74.9%) 365 (68.9%) 0.037 Myocardial infarction255 (48.3%) 296 (56.0%) 242 (45.7%) 0.003 Hypertension 424 (79.8%) 422(79.6%) 428 (80.8%) 0.89 Stroke or PVD 78 (14.7%) 102 (19.3%) 117(22.2%) 0.007 Anemia 174 (36.3%) 216 (47.2%) 238 (50.6%) <0.001 Thyroiddisease 60 (11.3%) 61 (11.5%) 57 (10.8%) 0.92 Depression 28 (5.3%) 41(7.7%) 35 (6.6%) 0.27 Hyperlipidemia 298 (56.1%) 267 (50.5%) 244 (46.0%)0.004 Current smoker 92 (17.4%) 114 (21.6%) 105 (19.8%) 0.23 COPD orasthma 106 (20.0%) 102 (19.2%) 112 (21.2%) 0.73 Diabetes mellitus 247(46.5%) 229 (43.2%) 258 (48.7%) 0.2 History of Atrial 260 (49.2%) 291(55.0%) 310 (58.8%) 0.007 Fibrillation/Flutter AICD 73 (13.7%) 106(20.0%) 68 (12.8%) 0.002 Biventricular pacemaker 54 (10.2%) 60 (11.3%)47 (8.9%) 0.41 Pacemaker 50 (9.5%) 66 (12.5%) 65 (12.3%) 0.22 MedicationBeta-blockers 417 (78.7%) 396 (74.7%) 385 (72.6%) 0.068 ACEIs 353(66.6%) 312 (58.9%) 330 (62.3%) 0.033 ARBs 85 (16.0%) 85 (16.0%) 71(13.4%) 0.38 ACE-I/ARB 424 (80.0%) 390 (73.6%) 393 (74.2%) 0.026 MRA 264(49.8%) 226 (42.6%) 229 (43.2%) 0.033 Digoxin 159 (30.0%) 153 (28.9%)146 (27.5%) 0.68 Nitrates 136 (25.7%) 146 (27.6%) 135 (25.5%) 0.68Hydralazine 17 (3.2%) 15 (2.8%) 13 (2.5%) 0.76 CCBs 74 (14.0%) 71(13.4%) 81 (15.3%) 0.67 Signs and symptoms Orthopnea 445 (84.3%) 440(84.5%) 435 (82.5%) 0.65 Dyspnea at rest (NYHA IV) 264 (50.6%) 304(59.1%) 327 (64.8%) <0.001 Angina pectoris 134 (25.2%) 117 (22.1%) 111(21.0%) 0.24 CCS Class III & IV 48 (36.4%) 38 (33.0%) 29 (26.6%) 0.27Edema 82 (15.4%) 151 (28.5%) 184 (34.8%) <0.001 Jugular venousdistension 190 (39.7%) 195 (40.5%) 199 (41.6%) 0.82 Edema & raised JVP127 (26.5%) 148 (30.7%) 150 (31.4%) 0.2 Rales 37 (7.0%) 55 (10.4%) 62(11.7%) 0.027

The association of IL-6 levels and diuretic response is shown in Table5, below. The diuretic response was defined as weight change on day 4per 40 mg of furosemide (or equivalent doses) administered from baselineto day 3.

TABLE 5 Diuretic Response Beta p-value Univariable 0.06 0.027 Model 1(age, sex) 0.06 0.030 Model 2(model 1 + eGFR, BMI) 0.06 0.035

Table 6 and FIGS. 7A and 7B show the association of IL-6 levels withall-cause mortality at 180 days and the association of IL-6 levels withall-cause mortality at 60 days and/or cardiovascular relatedrehospitalization (CV hosp).

TABLE 6 Cox Regression Results All-cause mortality at 60 days mortality180 days and/or CV hosp Univariable 1.59 (1.43-1.76) <0.001 1.14(1.04-1.26) 0.007 Model 1 1.57 (1.41-1.74) <0.001 1.14 (1.04-1.26) 0.007Model 2 1.55 (1.39-1.72) <0.001 1.11 (1.00-1.22) 0.049 Model 3 1.50(1.35-1.68) <0.001 1.09 (0.99-1.21) 0.087 PROTECT model 1.41 (1.26-1.58)<0.001 1.06 (0.95-1.17) 0.309 Model 1: age, sex Model 2: model 1 + eGFR,BMI Model 3: model 2 + BNP PROTECT model: age, previous hospitalizationfor HF, edema at admission, sodium, bun (log), creatinine (log) andalbumin

Table 7 and FIGS. 8A and 8B show that an increase in IL-6 levels at day7 compared to the baseline predicts adverse outcomes.

TABLE 7 Cox Regression Results >1 pg/mL <1.0 pg/mL >1.0 pg/mL decreaseincrease/decrease increase 180 days all-cause mortality Univariable 2.13(1.16-3.91) 0.014 ref  3.01 (1.65-5.49) <0.001 Model 1 1.79 (0.96-3.32)0.066 ref 2.80 (1.53-5.11) 0.001 Model 2 1.80 (0.95-3.44) 0.073 ref 2.65(1.41-4.97) 0.002 Model 3 1.65 (0.87-3.16) 0.128 ref 2.35 (1.25-4.44)0.008 PROTECT 1.71 (0.90-3.26) 0.101 ref 2.38 (1.30-4.47) 0.007 60 daysmortality and/or CV hosp Univariable 1.31 (0.91-1.92) 0.146 ref 1.83(1.26-2.65) 0.001 Model 1 1.31 (0.89-1.92) 0.002 ref 1.81 (1.25-2.62)0.002 Model 2 1.29 (0.88-1.92) 0.187 ref 1.70 (1.16-2.48) 0.006 Model 31.28 (0.86-1.89) 0.223 ref 1.67 (1.14-2.45) 0.008 PROTECT 1.20(0.81-1.78) 0.353 ref 1.57 (1.07-2.30) 0.020 Model 1: age, sex Model 2:model 1 + eGFR, BMI Model 3: model 2 + BNP PROTECT model: age, previoushospitalization for HF, edema at admission, sodium, bun (log),creatinine (log) and albumin

8.8.6. Example 6: Association of IL-6 Levels with Outcomes in aWorsening Heart Failure Population

Methods.

Data from the BIOlogy Study to TAilored Treatment in Chronic HeartFailure (BIOSTAT-CHF) study was analyzed to investigate the associationof IL-6 with outcomes in patients with worsening heart failure. Inbrief, BIOSTAT-CHF was a multicenter, multinational, prospective,observational study including 2516 patients from 69 centers in 11European countries (Voors et al., 2016, Eur. J. Heart Fail. 18:716-726).We performed secondary analyses in the BIOSTAT-CHF study, excludingpatients with ferritin <100 from subsequent analysis. Inclusion criteriafor the index cohort include: patients with >18 years of age, havingsymptoms of new-onset or worsening HF, confirmed either by a LVEF of≤40% or BNP and/or NT-proBNP plasma levels >400 pg/ml or >2,000 pg/ml,respectively. Furthermore, these patients had not been previouslytreated with an ACEi/ARBs and/or beta-blocker or they were receiving≤50% of the target doses of these drugs at the time of inclusion andanticipated initiation or up-titration of ACEi/ARBs and beta-blockers.All patients needed to be treated with loop diuretics.

TABLE B Inclusion criteria Age >18 Diagnosed with HF Previous documentedadmission with HF requiring diuretic treatment Treated withfurosemide >20 mg/day or equivalent Not previously treated or receiving<50% of target doses of ACE inhibitors/ARBs and/or beta-blockersaccording to 2008 ESC guidelines Anticipated uptitration of ACEinhibitors/ARBs and/or beta-blockers

In total, IL-6 was measured in 2329 patients with worsening HF from theBIOSTAT-CHF study. The primary outcome of this study was a compositeoutcome of all-cause mortality and hospitalization for HF.

Statistical Analyses.

Baseline characteristics are presented according to tertiles of IL-6.Differences between tertiles of baseline characteristics were testedusing one-way analysis of variance (ANOVA), Kruskal Wallis or chi2-testwhere appropriate. Survival analysis was performed using Cox regressionanalysis correcting for clinically relevant variables and theBIOSTAT-CHF risk model. The BIOSTAT-CHF risk model for all-causemortality and/or hospitalization for heart failure includes: age,N-terminal pro-B-type natriuretic peptide (NT-proBNP), hemoglobin (Hb),the use of a beta-blocker at time of inclusion, a HF-hospitalization inyear before inclusion, peripheral edema, systolic blood pressure,high-density lipoprotein cholesterol and sodium (Voors et al., 2017,Eur. J. Heart Fail. 19:627-634). We performed interaction analysisbetween ferritin levels and IL-6 for the primary outcome. To investigatethe association of IL-6 with outcomes depending on the position of theTMPRS6 SNP (rs855791).

Results.

The baseline characteristics of the population are described in Table 8,below. Higher levels of IL-6 at baseline are associated with higherlevels of NTproBNP and Anemia (FIG. 9).

TABLE 8 Baseline Characteristics Factor 1st tertile 2nd tertile 3rdtertile 781 775 773 N 0.3-3.4 pg/mL 3.5-7.8 pg/mL 7.9-260.7 pg/mLp-value Demographics Age 66.3 (12.2) 69.3 (11.7) 70.9 (11.7) <0.001Female 200 (25.6%) 200 (25.8%) 212 (27.4%) 0.67 HF status HFrEF 607(84.1%) 568 (81.6%) 524 (78.3%) <0.001 HFmrEF 92 (12.7%) 74 (10.6%) 87(13.0%) HFpEF 23 (3.2%) 54 (7.8%) 58 (8.7%) BMI 27.9 (5.1) 27.7 (5.6)27.7 (5.6) 0.77 Ischemic etiology 329 (43.2%) 353 (46.4%) 357 (46.7%)0.31 NYHA I 70 (9.0%) 82 (10.6%) 62 (8.0%) 0.005 II 400 (51.2%) 355(45.8%) 320 (41.4%) III 199 (25.5%) 216 (27.9%) 251 (32.5%) IV 22 (2.8%)25 (3.2%) 33 (4.3%) NA 90 (11.5%) 97 (12.5%) 107 (13.8%) Systolic BP126.4 (20.0) 124.3 (22.4) 123.7 (23.5) 0.034 Diastolic BP 76.7 (12.7)74.8 (13.4) 73.5 (14.0) <0.001 LVEF 30.6 (8.9) 30.9 (11.1) 31.3 (11.7)0.54 Heart Rate 76.4 (18.2) 80.1 (18.9) 83.6 (21.0) <0.001 Signs andsymptoms Peripheral edema Not Present 352 (57.2%) 253 (39.7%) 180(26.4%) <0.001 Ankle 170 (27.6%) 188 (29.5%) 221 (32.5%) Below Knee 83(13.5%) 156 (24.5%) 194 (28.5%) Above Knee 10 (1.6%) 40 (6.3%) 86(12.6%) Elevated JVP No 420 (76.5%) 318 (58.9%) 289 (53.7%) <0.001 Yes106 (19.3%) 190 (35.2%) 216 (40.1%) Uncertain 23 (4.2%) 32 (5.9%) 33(6.1%) Hepatomegaly 75 (9.6%) 128 (16.6%) 132 (17.1%) <0.001 Orthopnea168 (21.6%) 273 (35.3%) 361 (46.8%) <0.001 Medical history Anemia 158(23.6%) 271 (38.1%) 348 (47.0%) <0.001 Atrial fibrillation 293 (37.5%)372 (48.0%) 387 (50.1%) <0.001 Diabetes mellitus 208 (26.6%) 270 (34.8%)276 (35.7%) <0.001 COPD 113 (14.5%) 126 (16.3%) 163 (21.1%) 0.002 Renaldisease 152 (19.5%) 229 (29.5%) 261 (33.8%) <0.001 Hypertension 486(62.2%) 496 (64.0%) 473 (61.2%) 0.51 Peripheral artery 60 (7.7%) 92(11.9%) 105 (13.6%) <0.001 disease Stroke 59 (7.6%) 84 (10.8%) 76 (9.8%)0.075 PCI 163 (20.9%) 166 (21.4%) 173 (22.4%) 0.76 CABG 120 (15.4%) 122(15.7%) 156 (20.2%) 0.02 Medication Loop diuretics 779 (99.7%) 769(99.2%) 769 (99.5%) 0.36 ACE/ARB 601 (77.0%) 564 (72.8%) 518 (67.0%)<0.001 Beta blocker 675 (86.4%) 654 (84.4%) 603 (78.0%) <0.001Aldosterone 446 (57.1%) 411 (53.0%) 382 (49.4%) 0.01 antagonistLaboratory Hemoglobin 13.7 (1.7) 13.2 (1.8) 12.7 (2.0) <0.001 Totalcholesterol 4.5 (3.7, 5.5) 4.1 (3.4, 4.9) 3.7 (3.1, 4.5) <0.001 AST 24.0(19.0, 32.0) 25.0 (19.0, 34.0) 27.0 (20.0, 39.0) <0.001 ALT 25.0 (18.0,37.0) 25.0 (17.0, 37.0) 23.5 (15.0, 40.0) 0.12 Sodium 140.0 (138.0,142.0) 140.0 (137.0, 142.0) 139.0 (136.0, 141.0) <0.001 Potassium 4.3(4.0, 4.6) 4.2 (3.9, 4.6) 4.2 (3.8, 4.6) <0.001 HbA1c 6.0 (5.6, 6.7) 6.5(5.9, 7.5) 6.5 (5.9, 7.3) <0.001 NT-proBNP 2661.0 (1445.0, 4820.0)4344.0 (2517.0, 7837.0) 5734.5 (3141.0, 11452.0) Troponin-I 0.0 (0.0,0.1) 0.0 (0.0, 0.1) 0.0 (0.0, 0.1) <0.001

As shown in Table 9 and FIGS. 10A and 10B, levels of IL-6 measured atbaseline were associated with the combined outcome of all-causemortality and/or a hospitalization for HF as well as all-cause mortalityalone at two years.

TABLE 9 Cox Regression Analyses All-cause mort and/or HF All-causemortality hosp at 2 years at 2 years HR (95% CI) p-value HR (95% CI)p-value Univariable 1.38 (1.31-1.46) <0.001 1.42 (1.32-1.53) <0.001Model 1 1.34 (1.26-1.42) <0.001 1.48 (1.38-1.58) <0.001 Model 2 1.25(1.17-1.33) <0.001 1.34 (1.24-1.44) <0.001 Model 3 1.24 (1.16-1.32)<0.001 1.33 (1.23-1.44) <0.001 BIOSTAT model 1.13 (1.04-1.19) 0.001 1.20 (1.11-1.31) <0.001 Model 1: Age, sex Model 2: model 1 + BMI,country, hypertension (history of), diabetes (history of) and anemiaModel 3: model 2 + beta-blocker at baseline, ACEi/ARB at baseline andMRA at baseline BIOSTAT model: age, N-terminal pro-B-type natriureticpeptide (NT-proBNP), hemoglobin (Hb), the use of a beta-blocker at timeof inclusion, a HF-hospitalization in year before inclusion, peripheraledema, systolic blood pressure, high-density lipoprotein cholesterol andsodium

As shown in Table 10 and FIG. 11, there is no differential associationof IL-6 with outcome depending on ferritin status.

TABLE 10 Cox Regression Analyses Ferritin >100 ug/L HR (95% CI) p-valueInteraction ferritin *IL-6 Univariable 1.39 (1.27-1.52) <0.001 0.610Model 1 1.35 (1.23-1.48) <0.001 Model 2 1.26 (1.14-1.40) <0.001 Model 31.26 (1.14-1.39) <0.001 BIOSTAT model 1.11 (0.99-1.22) 0.052  Model 1:Age, sex Model 2: model 1 + BMI, country, hypertension (history of),diabetes (history of) and anemia Model 3: model 2 + beta-blocker atbaseline, ACEi/ARB at baseline and MRA at baseline BIOSTAT model: age,N-terminal pro-B-type natriuretic peptide (NT-proBNP), hemoglobin (Hb),the use of a beta-blocker at time of inclusion, a HF-hospitalization inyear before inclusion, peripheral edema, systolic blood pressure,high-density lipoprotein cholesterol and sodium

As shown in Table 11, there was no differential association of IL-6 withoutcome depending on TMPRSS6 genotype.

TABLE 11 IL-6 according to TMPRSS6 allele TMPRSS6 HR (95% CI) p-value AA(n = 191) 1.50 (1.19-1.89) 0.001  AG (n = 579) 1.34 (1.17-1.54) <0.001GG (n = 409) 1.54 (1.32-1.79) <0.001

9. INCORPORATION BY REFERENCE

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

10. EQUIVALENTS

While this invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

What is claimed is:
 1. A method of treating left ventricular heartfailure, the method comprising: administering to a patient with leftventricular heart failure a therapeutically effective amount of an antiIL-6 antibody.
 2. The method of claim 1, wherein the patient haselevated pre-treatment plasma IL-6 levels.
 3. The method of claim 2,wherein the patient has a pre-treatment plasma IL-6 level of greaterthan 2 pg/mL.
 4. The method of claim 1, wherein the patient has adiuretic efficiency of less than 500 mmol Na/doubling of loop diureticdose.
 5. The method of claim 4, wherein the patient has a diureticefficiency of less than 200 mmol Na/doubling of loop diuretic dose. 6.The method of claim 1, wherein the patient has diuretic resistant heartfailure.
 7. The method of claim 1, wherein the patient has acute heartfailure.
 8. The method of claim 1, wherein the patient has chronic heartfailure.
 9. The method of claim 1, wherein the anti-IL-6 antibodycomprises all six variable region CDRs of MEDI5117.
 10. The method ofclaim 9, wherein the antibody comprises the VH and VL of MEDI5117. 11.The method of claim 10, wherein the antibody is MEDI5117.
 12. The methodof claim 1, further comprising administering a diuretic.
 13. The methodof claim 1, wherein a single species of antibody is administered,without administration of any other antibody species.
 14. The method ofclaim 1, wherein the anti-IL-6 antibody is MED5117; and wherein MED5117is administered subcutaneously in a flat dose of from 10-20 mg.